In 1850, the worsening economic situation in Ireland led his family to emigrate to Canada, where he settled in Seymour Township, Ontario and began farming.

Although his formal education was slight, his knowledge and dedication to field work became sufficiently advanced that he gained the notice and respect of several professional botanists.

By 1860 he was teaching school in Belleville, and had established correspondence with botanists such as Asa Gray, Sir William Jackson Hooker, George Lawson, and Louis-Ovide Brunet.

Since Macoun's travels corresponded to a time of unusually high rainfall, he concluded that large regions of the Northwest were ideally suited to agriculture.

In concert with the political consideration of forestalling northwards American expansion, Macoun's assessment contributed much to the final southern routing of the CPR across the prairies.

Every summer was dedicated to fieldwork, and for the remainder of his life Macoun was a prolific collector and cataloguer of Canadian flora and fauna, even after suffering a debilitating stroke in 1912.