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Mazzolini was born in Arcevia, in the Marche. He founded a nationalist group in Macerata, and soon became editor of the daily newspaper L'Unione. An active irredentist and advocate of Italy's entry into World War I, he was a volunteer soldier in 1915, and was awarded a War Merit Cross. In 1918, Mazzolini returned to Ancona and was deputy editor of L'Ordine newspaper, interrupting his assignment in order to join Gabriele D'Annunzio as the latter attempted to seize Fiume for an "unredeemed" Italy (1919).
A member of the provincial council in Ancona, he took part in the March on Rome of 1922, and a year later joined the Partito Nazionale Fascista (PNF) - becoming one of its leaders as of 1924-1925. A deputy secretary for the PNF, he contributed to its Propaganda Office, represented it in the Italian Chamber of Deputies from 1924 on, and was awarded a supervisory position as member of the Grand Council of Fascism (from 1926).
In quick succession, he renounced all political missions, and returned to journalism for a while, before beginning a career in diplomacy: he was Italy's envoy to Brazil, Uruguay, the British Mandate of Palestine and Egypt. After the start of World War II and the attack carried out by the Axis Powers on their former ally, the Kingdom of Yugoslavia, Serafino Mazzolini was High Commissioner for Italian-occupied Montenegro in 1941, and held a high-ranking position in the Ministry of Foreign Affairs in 1943.
After Mussolini's ousting and the Armistice of Cassibile (8 September 1943) between Italy and the Allies, Mazzolini joined the Nazi German-backed and Mussolini-led Italian Social Republic as its deputy-secretary for Foreign Affairs. Throughout this period, he led a modest life, and was forced to acquire large debts in order to procure medicine that promised to keep him alive. He died at San Felice del Benaco as the result of a septicemia produced by an insulin injection.
His elder brother, Conte Quinto Mazzolini, served as Italian consul in Jerusalem, and undertook negotiations with Abraham Stern, head of the Lehi terrorist group, which sought (but failed) to obtain Italian recognition of Jewish sovereignty in Palestine in exchange for placing Zionism under the aegis of Italian fascism.
- Joseph Heller, The Stern Gang: ideology, politics and terror, 1940-1949,Frank Cass 1995 p.78.