<p>Aboveground litter not only is an important nutrient source for soil microbes but also regulates the microclimate in topsoil, and consequently affects microbial activities and related ecological processes. Here, we used a three-year experiment manipulating litterfall inputs to soil in a coniferous–broadleaf mixed forest in Central China to assess how soil microbial community and respiration responded to litter-derived carbon (C) change. Our results showed that litter addition increased gram-positive (GP) bacteria abundance by increasing soluble C content, decreased fungi abundance by increasing the ratio of dissolved organic carbon to available nitrogen and decreasing soil pH, but had no effect on gram-negative (GN) bacteria abundance. Litter exclusion decreased GP abundance but had little effect on GN and fungi abundance. Our study highlights the crucial roles of the ratio of substrate carbon to nitrogen in regulating bacterial and fungal communities and soil pH in regulating fungal communities. The shifts in microbial communities have crucial effects on microbial respiration. Litter addition increased microbial respiration by enhancing GP abundance and decreasing fungal abundance, while litter exclusion decreased microbial respiration by reducing GP abundance. Our findings suggest that identifying and describing soil dissolved organic carbon are key for understanding microbial community composition because of their significant relationships with ecosystem C emission processes.</p>