Soil physicochemical protection, substrates, and microorganisms are thought to modulate the temperature sensitivity of soil carbon decomposition (Q10), but their regulatory roles have yet to be distinguished because of the confounding effects of concurrent changes of them. Here, we sought to differentiate these effects through microorganism reciprocal transplant and aggregate disruption experiments using soils collected from seven sites along a 5000-km latitudinal transect encompassing a wide range of climatic conditions and from a 4-year laboratory incubation experiment. We found direct microbial regulation of Q10, with a higher Q10 being associated with greater fungal:bacterial ratios. However, no significant direct effects of physicochemical protection and substrate were observed on the variation in Q10 along the latitudinal transect or among different incubation time points. These findings highlight that we should move forward from physicochemical protection and substrate to microbial mechanisms regulating soil carbon decomposition temperature sensitivity to understand and better predict soil carbon-climate feedback.