AbstractSeasonality is a key feature of the biosphere and the seasonal dynamics of soil carbon (C) emissions represent a fundamental mechanism regulating the terrestrial–climate interaction. We applied a microbial explicit model—CLM‐Microbe—to evaluate the impacts of microbial seasonality on soil C cycling in terrestrial ecosystems. The CLM‐Microbe model was validated in simulating belowground respiratory fluxes, that is, microbial respiration, root respiration, and soil respiration at the site level. On average, the CLM‐Microbe model explained 72% (n = 19, p < 0.0001), 65% (n = 19, p < 0.0001), and 71% (n = 18, p < 0.0001) of the variation in microbial respiration, root respiration, and soil respiration, respectively. We then compared the model simulations of soil respiratory fluxes and soil organic C content in top 1 m between the CLM‐Microbe model with (CLM‐Microbe) and without (CLM‐Microbe_wos) seasonal dynamics of soil microbial biomass in natural biomes. Removing soil microbial seasonality reduced model performance in simulating microbial respiration and soil respiration, but led to slight differences in simulating root respiration. Compared with the CLM‐Microbe, the CLM‐Microbe_wos underestimated the annual flux of microbial respiration by 0.6%–32% and annual flux of soil respiration by 0.4%–29% in natural biomes. Correspondingly, the CLM‐Microbe_wos estimated higher soil organic C content in top 1 m (0.2%–7%) except for the sites in Arctic and boreal regions. Our findings suggest that soil microbial seasonality enhances soil respiratory C emissions, leading to a decline in SOC storage. An explicit representation of soil microbial seasonality represents a critical improvement for projecting soil C decomposition and reducing the uncertainties in global C cycle projection under the changing climate.