AbstractCO2 efflux from the litter layer, litter heterotrophic respiration, is an important component of the forest carbon cycle. Litter heterotrophic respiration mediated by microorganisms varies in response to seasonal environmental changes, such as temperature and moisture. Here, we aimed to quantify seasonal variation in litter heterotrophic respiration and determine how the microbial biomass influences microbial activity and hence litter heterotrophic respiration in a warm temperate forest. We performed in situ high‐frequency measurements of litter heterotrophic respiration per unit area (R_area), which are able to capture CO2 pulses during rainfall, for over 2 years. Microbial activity, which is the CO2 efflux per unit weight (R_mass) considering the change in the amount of substrate, was calculated based on R_area. In parallel, we measured substrate‐induced respiration (SIR) each month, as an index of microbial biomass. We identified seasonal hysteresis in R_area, which was higher in spring (January to July) than in fall (August to December), despite the temperature being similar in both periods. Of interest, R_mass and SIR also showed similar seasonal hysteresis in relation to temperature. Additionally, potential microbial activity without the effect of temperature and moisture was positively related to SIR. This result indicates that seasonal hysteresis with temperature in microbial activity was driven by microbial biomass seasonality, and thus, it leads to seasonal hysteresis in litter heterotrophic respiration in relation to temperature. Our findings highlight the importance of not only the environmental factors and substrate depletion but also biotic factors for estimating heterotrophic respiration.