Microbial methane production (methanogenesis) is responsible for more than half of the annual emissions of this major greenhouse gas to the atmosphere. Although the stable isotopic composition of methane is often used to characterize its sources and sinks, strictly empirical descriptions of the isotopic signature of methanogenesis currently limit these attempts. We developed a metabolic-isotopic model of methanogenesis by carbon dioxide reduction, which predicts carbon and hydrogen isotopic fractionations, and clumped isotopologue distributions, as functions of the cell’s environment. We mechanistically explain multiple isotopic patterns in laboratory and natural settings and show that these patterns constrain the in situ energetics of methanogenesis. Combining our model with data from environments in which methanogenic activity is energy-limited, we provide predictions for the biomass-specific methanogenesis rates and the associated isotopic effects.