After graduating from the University of Athens in 1927, he went to the Sorbonne, which at the time was a major center of Byzantine studies with scholars like Charles Diehl and Ferdinand Lot.
His first major work was a detailed study of the late Byzantine Despotate of the Morea, published in French (Le despotat grec de Morée (1262–1460)) in two volumes, one in 1932 and the other, delayed by World War II, in 1953.
The Academy of Athens elected him as a full member in 1966, and Zakythinos went on to serve as its president in 1974.
In 1971–76 he served as chairman of the International Association of Byzantine Studies (AIEB), and thereafter as its honorary president.
Zakythinos also served briefly as Minister to the Prime Minister in the interim 1963–64 government of Ioannis Paraskevopoulos, while after the fall of the Regime of the Colonels, he was elected to the Greek Parliament in the November 1974 elections, on the list of the conservative New Democracy party, serving until 1977.