He began his acting career in the Yiddish Theater District in 1913 and made his Broadway debut in 1915. He made his film debut in a minor uncredited role in 1916; in 1923 he made his named debut as E. G. Robinson in The Bright Shawl. One of many actors who saw his career flourish in the new sound film era rather than falter, he made only three films prior to 1930 but left his stage career that year and made 14 films between 1930–1932.

Robinson made 101 films in his fifty-year career. An acclaimed performance as the gangster Caesar Enrico "Rico" Bandello in Little Caesar (1931) led to him being typecast as a "tough guy" for much of his early career in works such as Five Star Final (1931), Smart Money (1931; his only movie with James Cagney), Tiger Shark (1932), Kid Galahad (1937) with Bette Davis and Humphrey Bogart, and A Slight Case of Murder. Due to age, he could not qualify for military service during World War II. In the 1940s, Robinson demonstrated his ability to succeed in comedic and film noir roles, including Raoul Walsh's Manpower (1941) with Marlene Dietrich and George Raft, Larceny, Inc. (1942) with Jane Wyman and Broderick Crawford, Billy Wilder's Double Indemnity (1944) with Fred MacMurray and Barbara Stanwyck, Fritz Lang's The Woman in the Window (1944) with Joan Bennett and Raymond Massey, Fritz Lang's Scarlet Street (1945) with Joan Bennett and Dan Duryea, and Orson Welles' The Stranger (1946) with Orson Welles and Loretta Young. He appeared for director John Huston as gangster Johnny Rocco in Key Largo (1948), the last of five films he made with Humphrey Bogart and the only one in which Bogart did not play a supporting role.

Robinson was an outspoken critic of fascism and Nazism, and donated more than US$ 250,000 to 850 political and charitable groups between 1939 and 1949. He was host to the Committee of 56 who gathered at his home on December 9, 1938, signing a "Declaration of Democratic Independence" which called for a boycott of all German Products. He played FBI agent Turrou in Confessions of a Nazi Spy, the first American film which showed Nazism as a threat to the United States in 1939, and Paul Ehrlich in Dr. Ehrlich's Magic Bullet and Paul Julius Reuter in A Dispatch from Reuter's, both Jewish Biography films of 1940. In 1942, he volunteered for military service and was sent to London.

On three occasions in 1950 and 1952, he was called to testify in front of the House Un-American Activities Committee (HUAC) and was threatened with blacklisting. Robinson took steps to clear his name, such as having a representative go through his check stubs to ensure that none had been issued to subversive organizations. He did not give names of Communist sympathizers, but he repudiated the organizations he had belonged to in the 1930s and 1940s and his own name was cleared, but he only received smaller roles and even those less frequently. Robinson "continued his ritual of rehabilitation by humiliation" in October 1952, when he wrote an article titled "How the Reds made a Sucker Out of Me", that was published in the American Legion Magazine. In spite of this, he was once again called to testify before the House Un-American Activities Committee in January 1954.

During this time, he appeared in numerous "B" movies for American International Pictures and other low-budget studios while attempting to regain his prior status as a major film actor. Some of these films, such as Vice Squad (1953) proved excellent in their own right, while others, such as 1958's Tank Battalion, were critical and commercial failures. Anti-communist director Cecil B. DeMille cast him as Dathan in The Ten Commandments in 1956, freeing him to make A-list films again. Robinson's acting career was subsequently bolstered by notable roles in 1959's A Hole in the Head opposite Frank Sinatra and The Cincinnati Kid (1965), which showcased Robinson alongside Steve McQueen.

Robinson's last-filmed scene of his last acting role was a euthanasia sequence in the science fiction cult film Soylent Green (1973). Immediately prior to filming the emotional scene, Robinson told his co-star, and longtime friend Charlton Heston that he was dying from cancer and had weeks to live, at best. Robinson died twelve days later.