MacNeill was born in Glenarm, County Antrim. He was educated in Belfast at the Royal University. MacNeill had an enormous interest in Irish history and immersed himself in the study of it. In 1893 he founded the Gaelic League with Douglas Hyde. He became editor of its newspaper - Gaelic Journal. In 1908 MacNeill was appointed professor of early Irish history at University College Dublin.
Through the Gaelic League MacNeill met members of Sinn Féin. He became chairman of the council that formed the Irish Volunteers in 1913. He later became chief of staff. MacNeill was vehemently opposed to the idea of an armed rebellion as he saw little hope of success. However, the IRB went ahead with its plans of an armed rebellion with the co-operation of James Connolly and the Citizen Army. Pádraig Pearse and some other Volunteer members supported this move also. Easter Sunday, April 23, 1916, was the day the revolution was to be staged. MacNeill heard about this the previous Thursday and agreed. However, on learning of the arrest of Roger Casement and the interception of German arms he ordered an immediated end to the rising. Pearse, Connolly and the others all agreed that they must go ahead with the rising - it began on Monday, April 24, 1916. After the surrender MacNeill was arrested and sentenced to life imprisonment.
MacNeill was released in 1917 and was elected MP for the National University of Ireland. In 1921 he supported the Anglo-Irish Treaty. Following this he became Minister for Education in the first government of the Irish Free State. In 1924 a Boundary Commission was set up to re-negotite the border between Northern Ireland and the Free State. MacNeill represented the Free State, however, he resigned after a report on its findings was published in a newspaper. In December 1925 the Free State government agreed with the British government that the boundary included the entire six counties. This angered many nationalists and MacNeill was the subject of much criticism. He was forced to resign as minister and he lost his Dáil seat in 1927.
He retired from politics completely and became Chairman of the Irish Manuscripts Commission. He published a number of books on Irish history. In his later years he devoted his life to scholarship.
Eoin MacNeill died in Dublin.