Son of a family of poor agricultural day laborers, as an autodidact, Di Vittorio became active in the socialist movement from adolescence: at fifteen, he was a member of the Socialist Youth Circle in Cerignola, and, in 1911, moved one to lead the Camera del Lavoro in Minervino Murge, and then the one in Bari.
As a native of the Mezzogiorno, Di Vittorio became involved in the syndicalist plans for solving the region's acute problems (in the manner illustrated by the Fasci Siciliani in final decade of the 19th century). A partisan of insurgence, Di Vittorio became a leader of the anarcho-syndicalist trade union Unione Sindacale Italiana after its formation in 1912. Unlike the majority of the group (which opposed militarism and Italy's entry into World War I), Di Vittorio, Alceste De Ambris, and Filippo Corridoni advocated irredentism. He subsequently fought in the conflict, and was discharged after being gravely wounded.
In 1921, after the Italian Socialist Party's split at its Congress in Livorno, he joined the Italian Communist Party (PCI). Di Vittorio joined the militant anti-fascist organization Arditi del Popolo, and was then elected to the Italian Chamber of Deputies on the PCI list in 1924. The new situation after the rise of Fascism and the March on Rome made him an enemy of Benito Mussolini's regime. Sentenced to twelve years in prison by a fascist special tribunal in 1925, he managed to flee to France, where he refounded the dissolved Confederazione Generale Italiana del Lavoro (CGIL) and led it into the Soviet-managed Profintern. Di Vittorio lived in the Soviet state from 1928 to 1930, representing Italy to the Red Peasant International. He then returned to Paris, where he entered the Politburo of the PCI.
He joined the Republican side fighting Francisco Franco's forces during the Spanish Civil War in 1936. He was Political Commissar of the XI International Brigade. After the fall of the Republic, he headed the board of a Paris-based newspaper with an anti-fascist message. After the World War II Fall of France to Nazi Germany, Di Vittorio was taken in custody by the Italian police, and detained on Ventotene. In 1943, as the Fascist regime fell in most of Italy, he was set free by partisans, and subsequently joined the Resistance in fighting against Mussolini's Italian Social Republic in Northern Italy.
When war ended in 1945, he was elected secretary of the CGIL - which he had helped bring back into politics through a pact he had signed the previous year with Dino Grandi and Oreste Lizzardi in Rome. The pact recreated CGIL as a representative of all forms of trade unionism - communist, socialist, Roman Catholic, and anarcho-syndicalist. In 1948, the group split after the communists organized a general strike to protest an assassination attempt on PCI-leader Palmiro Togliatti: catholics left to form Confederazione Italiana Sindacati Lavoratori. On March 5 1950, the social-democrats (who would become supporters of the Italian Democratic Socialist Party) took a similar attitude, and founded Unione Italiana del Lavoro.
He was followed in his position at the CGIL by Agostino Novella.