Born in San Martino di Lupari, Carlo Agostini studied at the seminary in Treviso, and was ordained to the priesthood by Bishop Andrea Longhin on 24 September 1910.

Agostini was a professor (1913–1925) and the rector (1925–1932) of the Treviso seminary, and was raised to the rank of privy chamberlain of his holiness in 1925.

Pope Pius XII announced on 29 November 1952 that he would elevate Agostini and twenty-three others to the College of Cardinals.

Agostini was initially buried in S. Michele cemetery, but his remains were later transferred to the crypt of St Mark's Basilica in November 1957.

The unexpected vacancy in Venice opened the way for the elevation of Angelo Giuseppe Roncalli, the future Pope John XXIII.