Globally rising soil microbial respiration (Rm) is a key process controlling the soil-to-atmosphere CO2 flux, yet its spatial variation and underlying mechanism at different scales is still poorly understood. A novel experiment based on the annual mean temperature of soil origin sites along a 4,200 km north–south transect of China forests revealed a hump-shaped relationship between Rm and latitude with a latitudinal threshold of 32.5°N. Microbial variables were more important in shaping Rm’ spatial variation at the continental scale than at the regional scales, but soil physicochemical property had comparably unique importance at different scales. Labile organic C was the most important factor in regulating the Rm’s variation at the continent and in the latitude > 32.5°N region, but fungi biomass was the most important factor in the latitude < 32.5°N region. Overall, our findings suggest different controlling factors of Rm’s variations on either side of the latitudinal threshold.