Methane (CH4) is an important greenhouse gas and amplifier of climate change. However, the causes of atmospheric CH4 variations over glacial-interglacial cycles remain unresolved. We propose that microbial methanogenesis along the shallow margins of sedimentary basins provides a source of atmospheric CH4 temporally connected with both advance and retreat of continental ice sheets. Extensive biodegradation of hydrocarbons in the Antrim Shale Formation, Michigan, United States, is associated with an active subsurface consortium of fermentative and methanogenic microorganisms. This activity was initially stimulated when saline formation waters were diluted by meltwater derived from overriding Pleistocene ice sheets. During glaciation, CH4 produced by this community accumulated in the shale at a rate of 1 Tg CH4 per 1000 yr as a result of ice coverage and increased hydrostatic pressure. We estimate that at present the Antrim Shale contains only 12%–25% of the cumulative mass of CH4 generated in the shale over the Pleistocene, indicating that CH4 that had accumulated during glaciation was subsequently released following ice-sheet retreat. While release from the Antrim Shale represents only a small part of the global CH4 budget, when extended to other glaciated sedimentary basins, subsurface methanogenesis may generate a substantial, previously unrecognized source of atmospheric CH4 during deglaciation.