Glaciers expanded in the Northern Hemisphere during the middle Pliocene. However, whether they extended into the midlatitude lowlands remains unknown. A Pliocene sequence from a buried, deep bedrock trench in the midlatitude James Bay Lowland, Canada (52°49.5′N, 83°52.5′W), contains a till and an overlying lacustrine deposit rich in fossil pollen. Magnetostratigraphy together with pollen-derived biostratigraphy constrains it to a time span from 3.6 to 3.0 Ma. Based on multiple lines of evidence, we are able to prove the deposition of the till by an early ice sheet, and hence glaciation of the lowland at ca. 3.5 Ma (3.6–3.4 Ma). After glaciation, rapid warming permitted thermophilic trees now exotic to this area to grow, which include oak, sweetgum, and cypress. Furthermore, pollen analysis indicates alternating Carolinian deciduous and boreal evergreen forests under a climate that oscillated and cooled gradually during a prolonged postglacial period from 3.5 to 3.0 Ma.