We investigated spatial and temporal variability in the carbon stable isotope ratio (δ13C) of soil heterotrophic respiration in a deciduous needle-leaf forest in Japan for 3 years. We used high-precision isotope measurement coupled with a sampling system optimized for soil respiration to capture this variability under natural conditions. The limitations of chamber-based measurements combined with spatial variation created a representation error that prevented precise estimates of flux-weighted mean δ13C, but we could nonetheless characterize the δ13C variations intrinsic to heterotrophic respiration. In the absence of root respiration, δ13C exhibited significant seasonal variation, with a greater range than in previous models. In a root-exclusion plot, δ13C was lowest at high temperatures but showed a different seasonal course from that of CO2 efflux. A simple model explained the seasonal variation in δ13C using interpool differences in δ13C of decomposed organic matter, in the temperature dependence of decomposition rates, and in the seasonal changes in pool size. The characteristic seasonality of δ13C appears to be associated with the properties of the forest, including litterfall patterns.