After serving with the U.S. Army during the Korean War, he trained in acting at the Pasadena Playhouse and later co-founded the East West Players.

[10] Though he had an interest in dramatics, Iwamatsu did not believe an artistic career was financially viable, and enrolled in the Pratt Institute School of Architecture while working in his father's print shop.

[5][7][8][13] He then trained at the Pasadena Playhouse and adopted the mononym Mako, as he found most people had difficulty pronouncing his full name.

He was nominated for an Academy Award for Best Supporting Actor for his role as engine-room worker Po-Han in the film The Sand Pebbles (1966).

[8] His other roles include the Chinese contract laborer Mun Ki in the epic movie The Hawaiians (1970) starring Charlton Heston and Tina Chen; Oomiak, the Inuit guide, in Disney's The Island at the Top of the World (1974); Yuen Chung in the film The Killer Elite (1975) directed by Sam Peckinpah and starring James Caan, Robert Duvall, and martial artist Takayuki Kubota; the sorcerer Nakano in Highlander III: The Sorcerer; Jackie Chan's uncle/sifu in Chan's first American movie The Big Brawl (1980); the wizard Akiro opposite Arnold Schwarzenegger in the two Conan movies Conan the Barbarian and Conan the Destroyer; the confidant to Chuck Norris' rogue cop in the thriller An Eye for an Eye (1982); and the Japanese spy in the comedy Under the Rainbow.

In 1990, he had a minor role in the psychological thriller Pacific Heights along with Matthew Modine, Melanie Griffith, and Michael Keaton; Yoshida-san in Rising Sun; Mr. Lee in Sidekicks; Kanemitsu in RoboCop 3 (1993); and Kungo Tsarong in Seven Years in Tibet (1997).

He also appeared in some Japanese television dramas and films, such as Masahiro Shinoda's Owls' Castle and Takashi Miike's The Bird People in China.

In 1965, frustrated by the limited roles available to Asian-American actors, Mako and six others formed the East West Players theater company, first performing out of a church basement.

During the company's 1981 season, to coincide with the Commission on Wartime Relocation and Internment of Civilians' hearings on redress, Mako exclusively produced plays about the Japanese-American incarceration.

[15] Mako's landlord at the time, Jerry Orbach, was also nominated for his role in Chicago; both lost, however, to George Rose from the revival of My Fair Lady.

Mako appeared on the television series McHale's Navy nine times, playing Imperial Japanese officers, soldiers, and sailors.

He appeared as Lo Sing, challenging Bruce Lee's Kato character in The Green Hornet episode "The Preying Mantis".

Mako's biggest television role to date came in 1967 in The Time Tunnel, playing a sadistic soldier during the last months of World War II.

After Mako's death, Greg Baldwin replaced him as Aku in Samurai Jack and Iroh in Avatar: The Last Airbender and The Legend of Korra.