A man named Timothy Templeton (Tobey Maguire) tells a story through his imaginative point of view as his seven year old self (Miles Christopher Bakshi) who lives his days having fun with his parents, Ted (Jimmy Kimmel) and Janice (Lisa Kudrow), and wishes it to be just the three of them forever. However, one day, Tim is surprised when a business suit-wearing infant shows up in a taxi at his house and Ted and Janice proudly call him Tim's little brother. Tim is envious of the attention the baby is receiving, not to mention suspicious when the infant acts odd around him, but his parents, being blind to the baby's eccentric behavior, try to convince him that they will grow to love each other.
Soon, Tim learns that the baby can talk like an adult (Alec Baldwin) and he introduces himself as "The Boss Baby". Seeing an opportunity to be rid of him, Tim decides to record a conversation between him and other toddlers who are over at Tim's house for a meeting (under the guise of a playdate by the parents) to do something about how puppies are receiving more love than babies. The Boss Baby and the other infants catch Tim with the recording and after a chase scene throughout the backyard and the house, the tape is terminated after The Boss Baby threatens to tear up Tim's favorite stuffed animal, Lam-Lam. With no evidence to support him, Tim is subsequently grounded by his parents for his actions during the chase between him and the infants
The Boss Baby comes to Tim and has him to suck on a pacifier that transports them to Baby Corp, a place where infants with adult-like minds work to preserve infant love everywhere. They are virtual, so they cannot be seen or heard. The Boss Baby explains to Tim that he was sent on a mission to see why puppies are getting more love than infants. He has infiltrated Tim's residence because his parents work for Puppy Co., which is unleashing a new puppy on the day that employees take their children to work. The Boss Baby also explains that he stays intelligent by drinking a "Secret Baby Formula" which enables a baby to act like an adult. However, if a baby does not drink it after a period of time, he or she reverts to a regular baby. He hopes to receive a promotion after dealing with Puppy Co.'s new puppy, but when they overhear Boss Baby's boss threaten to fire him for not bringing in information, thereby stranding him at the Templetons, he and Tim agree to work together to keep that from happening.
After appearing to have patched things over, Tim's parents lift the grounding and take them to Puppy Co. for "take your child to work day". While there, they slip away and find what they think is the plans for a "Forever Puppy", but it turns out to be a trap set by founder Francis E. Francis (Steve Buscemi). They discover that Francis used to be the head of Baby Corp. (and Boss Baby's idol), but was forced out when it was discovered that his lactose intolerance kept the secret formula from working properly. Vowing revenge, Francis founded Puppy Corp. and intends to have the Forever Puppies overshadow babies by stealing Boss Baby's serum bottle and infecting the puppies with it. Tim's parents go with Francis to Las Vegas, and Francis has his brother pose as Tim and Boss Baby's babysitter to keep them from interfering.
Without a steady flow of serum to keep his intelligence in check, Boss Baby begins reverting back to being a normal baby. Despite this, he and Tim manage to evade the "babysitter" long enough to get to the airport, but are too late to intercept Tim's parents. Upset, Tim blames Boss Baby for using his family for his own ulterior motives, for which Boss Baby, after some hesitance, apologizes. After sneaking on a plane for Elvis impersonators bound for Vegas, they stall Francis' presentation when the brother (disguised as an Elvis impersonator) unwittingly gives away their plan.
Furious at their interference, Francis proceeds to lock Tim's parents up so he can burn them with exhaust from a rocket used to launch the Forever Puppies. Tim and Boss Baby fight with him, and then push him into the formula. Boss Baby opens the rocket to let the dogs out, so they can save Tim's parents. After he successfully does that, he returns to baby state while still on the rocket, but Tim sings to him with a family song to show his appreciation, causing him to jump off of the rocket before it launches. Francis, having reverted back to baby form, attempts to attacks them again, but his brother interferes, stating the he'll "raise him right this time" now that he's a baby again.
Boss Baby gets promoted, and Tim goes back to being an only child, but Tim and Boss Baby, having grown closer, start to miss each other. Boss Baby, fed up, decides to be part of the Templeton family. He returns to the Templeton family as a regular baby named Theodore Lindsey. Tim, now an adult, and now the father of an older daughter and an infant daughter, who acts exactly like Theodore did when he was Boss Baby.
- Christopher Robin, Weekend and Rainboom Superheroes of Justice, The 100 Acre Avatar League, Ash Ketchum, Pikachu, Serena, Misty, Togepi, Brock, Littlefoot, Cera, Ducky, Petrie, Spike, Chomper, Ruby, Guido, Grandpa Longneck, Grandma Longneck, Genie, Simba, Nala, Timon, Pumbaa, Sora, Riku, Kairi, Mickey Mouse, Donald Duck, Goofy, Pluto, SpongeBob SquarePants, Patrick Star, Squidward Tentacles, Sandy Cheeks, Eugene Krabs, The Mighty Morphin Power Rangers, Scooby-Doo, Shaggy Rogers, Fred Jones, Daphne Blake, Velma Dinkley, The DigiDestined and their Digimon, Kazuto Izuka, Narue Nanase, Masaki Maruo, Hajime Yagi, Kanaka Nanase, Miss Frizzle, Tim, Arnold, Dorothy Ann, Phoebe, Keesha, Wanda, Ralphie, Carlos, Mr. Peabody, Sherman, Penny Peterson, Serena and the Sailor Scouts, Bloom, Stella (Winx Club), Flora, Musa, Tecna, Layla (Winx Club), Laval, Cragger, Eris, Gorzan, Worriz, Razar, Bladvic, Rogon, The Bowser Family, Psycho Rangers,Dr. Facilier, Jafar, Maleficent, Myotismon, Hades, Nora Beady, The Grand Duke of Owls, Attila and Hun, Mr. Crocker, Vicky, Foop, Sheldon J. Plankton, Scar, The Crime Empire (Joker, Two-Face, Penguin, Negaduck, Megavolt, Quackerjack, Bushroot, and The Liquidator) Harley Quinn, The Mean Girls Gang (The Trix, Megan, The Dazzlings, Kurumi Tokisaki, Kyoko Kudo, Yuki Kashiwazaki, and Aya Kanazawa), Janet, Team Rocket guest stars appearances in this film.
- The Bowser Family, Psycho Rangers, Dr. Facilier, Jafar, Maleficent, Myotismon, Hades, Nora Beady, The Grand Duke of Owls, Attila and Hun, Mr. Crocker, Vicky, Foop, Sheldon J. Plankton, Scar, The Crime Empire (Joker, Two-Face, Penguin, Negaduck, Megavolt, Quackerjack, Bushroot, and The Liquidator) Harley Quinn, The Mean Girls Gang (The Trix, Megan, The Dazzlings, Kurumi Tokisaki, Kyoko Kudo, Yuki Kashiwazaki, and Aya Kanazawa), Janet, and Team Rocket will work with Francis E. Francis.
- Jim Cummings as Winnie the Pooh and Tigger
- Travis Oates as Piglet
- Tom Kenny as Rabbit
- Peter Cullen and Bud Luckey as Eeyore
- Jason Marsden as Tino Tonitini
- Grey DeLisle as Lor McQuarrie
- Phil LaMarr as Carver Descartes
- Kate Soucie as Tish Katsufrakis
LegoKyle14's Pooh's Adventure Team:
- Tara Strong as Timmy/Poof
- Daran Norris as Cosmo
- Susanne Blakeslee as Wanda
- Chris Hardwick as Otis
- Jeff Garcia as Pip
- Leigh-Allyn Baker as Abby,
- Tino Insana as Pig
- Cam Clarke as Freddy
- Rob Paulsen as Peck
- Grant Palmer as Linclon
- Catherine Taber as Lori
- Liliana Mumy as Leni
- Nika Futterman-Frost as Luna
- Cristina Pucelli as Luan
- Jesica DiCicco as Lynn/Lucy
- Grey DeLisle as Lana/Lola/Lily
- Lara Jill Miller as Lisa
- Ashleigh Ball as Rainbow Dash/Applejack
- Andrea Libman as Pinkie Pie/Fluttershy
- Tabitha St. Germain as Rarity
- Caitlin Murphy as Ronny Robinson
- Bob West as Barney
- Patty Wirtz as BJ
- Julie Johnson as Baby Bop
- Michaela Dietz as Riff
- Candace Cameron-Bure as D.J Tanner
Sonic876's Weekenders Adventure Team:
- Rebecca Shoichet as Sunset Shimmer
- Tara Strong as Sci-Twi
- Cathy Weseluck as Spike the Dog
- Josh Grelle as Shido
- Michelle Rojas as Tohka
- Bryn Apprill as Kotori
- Tia Ballard as Yoshino
- Michelle Lee as Origami
- Felecia Angelle as Mana
- Carly McKillip as Sakura Kinomoto
- Mona Marshall as Doraemon
- Johnny Yong Bosch as Noby Nobi
- Cassandra Morris as Sue/Leo and Luna
- Brian Beacock as Sneech
- Kaiji Tang as Takeshi "Big G" Goda