The Devonian Antrim Shale is an organic-rich, naturally fractured black shale in the Michigan Basin that serves as both a source and reservoir for natural gas. A well-developed network of major, through-going vertical fractures controls reservoir-scale permeability in the Antrim Shale. Many fractures are open, but some are partially sealed by calcite cements that retain isotopic evidence of widespread microbial methanogenesis. Fracture filling calcite displays an unusually broad spectrum of d 13 C values (þ34 to À41% PDB), suggesting that both aerobic and anaerobic bacterial processes were active in the reservoir. Calcites with high d 13 C values (>þ15%) record cementation of fractures from dissolved inorganic carbon (DIC) generated during bacterial methanogenesis. Calcites with low d 13 C values (<À32%) are solely associated with outcrop samples and record methane oxidation during cement precipitation. Fracture-fill calcite with d 13 C values between À10 and À30% can be attributed to variable organic matter oxidation pathways, methane oxidation, and carbonate rock buffering. Identification of 13 C-rich calcite provides unambiguous evidence of biogenic methane generation and may be used to identify gas deposits in other sedimentary basins.It is likely that repeated glacial advances and retreats exposed the Antrim Shale at the basin margin, enhanced meteoric recharge into the shallow part of the fractured reservoir, and initiated multiple episodes of bacterial methanogenesis and methanotrophic activity that were recorded in fracture-fill cements. The d 18 O values in both formation waters and calcite cements increase with depth in the basin (À12 to À4% SMOW, and þ21 to þ27% PDB, respectively). Most fracture-fill cements from outcrop samples have d 13 C values between À41 and À15% PDB. In contrast, most cement in cores have d 13 C values between þ15 and þ34% PDB. Radiocarbon and 230 Th dating of fracture-fill calcite indicates that the calcite formed between 33 and 390 ka, well within the Pleistocene Epoch.