The animals of Mufasa's kingdom arrive at Pride Rock to greet and honor the birth of his newborn son and their next king, Simba. Mufasa's brother, Scar, is enraged that he has lost his place as Mufasa's immediate successor upon the birth of the prince. Mufasa teaches his son the delicate balance of all living things and takes him through the Pride Lands, but warns him not to go outside the lands' limits, especially the elephant graveyard. Simba disobeys his father and is nearly killed by Scar's three hyena henchmen, but they are saved by Mufasa in the last minute. That night, Mufasa sternly warns Simba of the thin line between bravery and arrogance, and warmly teaches Simba that the great kings of the past are always up in the stars, watching over him and ready to guide him. Out of jealousy, Scar plots to kill his older brother and Simba, so that he could rule the Pride Lands. Scar's plot is partially fulfilled when he kills Mufasa after Mufasa saves Simba from a wildebeest stampede initiated by the hyenas under Scar's signal. Simba goes into exile believing Mufasa's death was his fault and years later, he encounters the wise mandrill Rafiki, who shows him that Mufasa is still alive inside Simba. To convince him further, Rafiki summons Mufasa's spirit, who tells Simba that he is the one true king and must take his place in the circle of life. Upon seeing his father again, Simba regains the confidence to face Scar for the throne.
Mufasa's role in the sequel is generally minor, ranging from his appearance in one of Simba's nightmares to his spirit's appearance in the sky at the beginning of the film, witnessing the welcoming ceremony of his granddaughter Kiara, and insisting on Rafiki encouraging her union with the outlander Kovu to reconcile the hostile prides despite Rafiki initially commenting that Mufasa's head is in the clouds after so long 'up there' for coming up with such an outrageous plan. Mufasa appears at the end of the film, praising Simba for his decision of letting the past go and allowing the banned lionesses to join the pride.
In the 2004 direct-to-video interquel The Lion King 1½, Mufasa is seen in three scenes: at the presentation of Simba, in the elephant graveyard on the way to save his son and Nala from the hyenas, and when his ghost is forming from the clouds above the grasslands at night.
Generally, Mufasa is an omniscient character whose wisdom and judgment is always correct, and to whom every character looks for guidance.
Prior to the events of the Lion King, Mufasa and Sarabi took in an orphaned lion cub and named him Jondugu (Johnny from Benny, Leo and Johnny's Adventures) out of love. After Kovu and Kiara inherited the Pridelands, and Simba and Nala went off to join Timon and Pumbaa on their adventures, Mufasa's spirit asked the wise old baboon Rafiki look after Jondugu and guided him to the Fantasy Adventure team.
- Mufasa is voiced by the acting veteran, James Earl Jones who also portrayed Darth Vader in Star Wars and Mr. Mertle in The Sandlot.
- Mufasa became one of the characters in Pooh's Adventures of The Lion King.
- Mufasa became one of the characters in Weekenders Adventures of The Lion King.
- Mufasa became one of the characters in Benny, Leo and Johnny's Adventures of The Lion King.
- Mufasa became one of the characters in SpongeBob's Adventures of The Lion King.
- Mufasa became one of the characters in The FT Squad's Adventures in The Lion King.
- Mufasa will become one of the characters in Littlefoot's Adventures of The Lion King.
- Mufasa will become one of the characters in Bloom's Adventures of The Lion King.
- Mufasa became one of the characters in Yogi Bear's Adventures of The Lion King.
- Mufasa will become one of the characters in Ash's Adventures of The Lion King II: Simba's Pride. In this movie, Mufasa will act as Ash's spirit guide into bringing the pridelanders and the outlanders together and stop the war.
- Mufasa became one of the characters in The Berenstain Bears' Adventures of the Lion King.
- Mufasa will become one of the characters in Dipper & Mabel meet The Lion King.
- Mufasa will become one of the characters in Sailor Moon's Adventures of The Lion King.