Shallow groundwaters were collected over and near buried kimberlites in the Attawapiskat River region of the James Bay Lowlands, Ontario, Canada in order to study the impact kimberlites have on CO 2 –CH 4 systematics. Groundwaters collected from boreholes in kimberlites and limestone, and from groundwaters in overlying Tyrell Sea sediment (TSS) were analyzed for δ 13 C DIC , δ 2 H H2O , δ 18 O H2O , dissolved inorganic carbon (DIC), and metal concentrations. Methane gas samples from borehole and TSS groundwaters were analyzed for concentration, δ 13 C CH4 , and δ 2 H CH4 . The CH 4 concentrations and Δ 13 C DIC–CH4 (isotope separation) values indicate biological carbonate reduction in TSS groundwaters overlying kimberlites. Whereas, Δ 13 C DIC–CH4 values from TSS groundwaters over limestone and from boreholes within limestone and kimberlite indicate the biological consumption of methane (oxidation). The δ 2 H H2O values from TSS over kimberlites are consistent with the variation in Δ 13 C DIC–CH4 , as they are less negative compared to where they should fall on the local meteoric water line, suggesting that methanogens are using lighter δ 2 H H2O values to produce CH 4 . Biological DIC reduction requires H + ions from H 2 O to form CH 4 . There is evidence in the water geochemistry to support the isotopic results, as the ratio of methane to calculated Fe 3+ (as amorphous Fe hydroxide), SO 4 2− , and O 2(aq) is largest in the majority of TSS groundwaters over kimberlites (where Δ 13 C DIC–CH4 values indicate CH 4 production). Low temperature serpentinization of olivine in kimberlite is not considered for CH 4 production, as redox conditions in kimberlite groundwaters do not support abiogenic methane production. The findings here suggest that kimberlites are indirectly influencing the CO 2 –CH 4 system by consuming oxidized ions in the overlying TSS, thereby creating a favorable environment for methane producing bacteria. In contrast, isotopes and geochemistry suggest methane oxidation in areas overlying limestone. The broader implication of this study is that variable lithology underlying sediment cover may impact biological methane production or consumption.