- Edward Asner as Carl Fredricksen (Jeremy Leary voiced Carl as a younger child). Docter and Rivera noted Asner's television alter ego, Lou Grant had been helpful in writing for Carl, because it guided them in balancing likable and unlikable aspects of the curmudgeonly character. When they met Asner and presented him with a model of his character, he joked, "I don't look anything like that." (The appearance of Carl is meant to resemble Spencer Tracy as he appeared in his final film, Guess Who's Coming to Dinner.) They tailored his dialogue for him, with short sentences and more consonants, which "cemented the notion that Carl, post-Ellie, is a disgruntled bear that's been poked awake during hibernation". In Colombia, unexpected publicity for the film was generated due to the uncanny similarity of Carl with Colombian ex-president Julio César Turbay Ayala.
- Christopher Plummer as Charles F. Muntz. Muntz is an old explorer looking for the beast of Paradise Falls; he vowed not to return to North America until he had captured the creature. He uses a group of dogs to aid him in his hunt. The name of his airship, Spirit of Adventure, may have been inspired by Charles Lindbergh's airplane, Spirit of St. Louis. In various interviews, Pete Docter has mentioned Howard Hughes and real life adventurers Charles Lindbergh and Percy Fawcett as inspirations for Muntz.
- Jordan Nagai as Russell. On their journey, Russell makes several comments to Carl that suggest that Russell's father and mother are no longer together. Russell's design was based on Pixar animator Peter Sohn. Docter auditioned 400 boys in a nationwide casting call for the part. Nagai, who is Japanese-American, showed up to an audition with his brother, who was actually the one auditioning. Docter realized Nagai behaved and spoke non-stop like Russell and chose him for the part. Nagai was 8 years old when cast. Docter encouraged Nagai to act physically as well as vocally when recording the role, lifting him upside down and tickling him for the scene where Russell encounters Kevin. Asian Americans have positively noted Pixar's first casting of an Asian lead character, in contrast to the common practice of casting non-Asians in Asian parts.
- Bob Peterson as Dug, a Golden Retriever who can talk. He is the misfit of a pack of talking dogs owned by Muntz. Peterson knew he would voice Dug when he wrote his line "I have just met you, and I love you," which was based on what a child told him when he was a camp counselor in the 1980s. The DVD release of the film features a short called Dug's Special Mission, which follows Dug just prior to his first meeting with Carl and Russell. Dug previously appeared in Ratatouille as a shadow on a wall that barks at Remy.
- Peterson also voices Alpha, a talking Doberman Pinscher and the leader of Muntz's pack of dogs. Pete Docter has stated that Alpha "thinks of himself as Clint Eastwood". Despite his menacing appearance, a frequent malfunction in Alpha's translating collar causes his voice to sound comically high-pitched and squeaky, as if he had been breathing helium. The normal voice for his translator is a resonant, intimidating bass. With both voices, Alpha has a roundabout speech pattern that causes his sentences to be longer than necessary.
- Pete Docter as Kevin, a large colorful prehistoric bird. Other than voicing Kevin, Docter also voices Campmaster Strauch, Russell's camp master, seen at the end of the film.
- Elizabeth Docter as Ellie Fredricksen as a younger child. The voice actor is the director's daughter, who also provided some of the drawings shown by Ellie.
- Delroy Lindo as Beta, a Rottweiler and one of Muntz's dogs.
- Jerome Ranft as Gamma, a Bulldog and one of Muntz's dogs.
- John Ratzenberger as Tom, a construction worker who asks if Carl is ready to sell his house.
- David Kaye as the newsreel announcer.
- UPisode One: Animal Calls - in the first episode, Russell demonstrates his ability to mimic animal calls.
- UPisode Two: First Aid - in the second episode, Russell tries to relieve a minor injury that Carl received.
- UPisode Three: Snipe Trap - in the third episode, Russell attempts to capture the elusive snipe.
Theme park attraction
- For a short time you could meet Carl, Russell and Dug, three characters from the film at Disney's Hollywood Studios at the Walt Disney World Resort. Their meeting location was in the Magic of Disney Animation Character Spot. Their former location currently houses Winnie the Pooh's Meet and Greet area. Since then, Russell and Dug have been moved to Animal Kingdom in Discovery Island. The Stars of "Up" also star in the new Pixar Pals Countdown to Fun Parade at Disney's Hollywood Studios.
- Russell and Dug are also part of the Redwood Creek Challenge Trail where guest take part in a fun filled challenge to earn a Wilderness Explorer Badge and to take part in the Senior Wilderness Explorer Ceremony at Grizzly Peak in Disney's California Adventures theme park.
- A scene from Up featuring Carl & Russell and a voice over commentary by Dug is shown as part of DCA's World of Color nighttime spectacular.