Methane generated by microorganisms is most often depleted in the doubly substituted isotopologue 12 CH 2 D 2 relative to the stochastic reference distribution. To constrain the controls on depleted ∆ 12 CH 2 D 2 values, we experimentally isolated the root cause with microorganisms that produce methane from methylphosphonate via the C-P lyase pathway. This mechanism of methane production preserves the three hydrogens from methylphosphonate and adds one hydrogen from water. When maintaining the same methylphosphonate source, but varying the D/H composition of growth medium water, we observed significant shifts in methane ∆ 12 CH 2 D 2 values, but little to no change in ∆ 13 CH 3 D values. We reproduced these observations with a model that considers only the combinatorial isotope effect. The variation in ∆ 12 CH 2 D 2 values of product methane resulted from the differences in D/H between reactants water and methylphosphonate. This work validates the hypothesis that combinatorial effects can strongly influence methane ∆ 12 CH 2 D 2 values, and must be considered for low temperature, abiotic or biotic systems where methane hydrogen is derived from multiple reservoirs.