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Holborn was born the son of Ludwig Holborn, the German physicist and "Direktor der Physikalisch-Technischen Reichsanstalt". In 19-- he became a student of Friedrich Meinecke at Berlin University, where he achieved a doctor of philosophy in 1924. After establishing at Heidelberg in 1926, he became Privatdozent there until he was called back to Berlin as Carnegie Professor of History and International Relationships at the private Deutsche Hochschule für Politik; there he worked until his dismissal in 1933.
To avoid the Nazi terror, that same year he fled to the United Kingdom, then emigrated to the United States in 1934. Shortly after coming to America, he was appointed guest professor of German history at Yale. He taught Diplomatic History at Tufts University, Mass., (1936–1942) and was a guest professor at the University of Vienna, Austria (1955). He became a U.S. citizen and during the Second World War he worked for the Office of Strategic Services as special assistant to the chief of its Research and Analysis Branch, William L. Langer. At the conclusion of the war he served as Randolph W. Townsend professor at Yale until 1959, when he was awarded the title of Sterling Professor of History at Yale University; here he continued to teach and write until his death in 1969.
In 1967 Holborn became the first president of the American Historical Association not born in the United States. Several specialists of German and European History in America, including Peter Gay, were students of Holborn.
Like their father, Hajo Holborn's children pursued successful careers in academic scholarship. His son Fred Holborn was a senior adjunct professor of American Foreign Policy at the School of Advanced International Studies at Johns Hopkins University before his death in 2005. Holborn's daughter, Hanna Holborn Gray (born 1930), is a historian of political thought in the Renaissance and Reformation. She is the Harry Pratt Judson Professor Emeritus at the University of Chicago and was the University's President for 15 years.
Prior to his emigration, Holborn was commissioned by the government to compose a history of the constitution of the Weimar Republic, resulting in the work "The Weimar Republic and the Birth of the German Democratic Party: The Hajo Holborn Papers, 1849-1956." Other works by Holborn include the History of Modern Germany series, spanning three volumes and covering a four-century period culminating in the capitulation of Hitler's regime in 1945.
Holborn's work has been praised by several of his distinguished peers (e.g. Fritz Stern).