Soil organic matter is one of the most important carbon (C) pools in terrestrial ecosystems, and future warming from climate change will likely alter soil C storage via temperature effects on microbial respiration. In this study, we collected forest soils from eight locations along a 3,700 km North–South transect in eastern China (NSTEC). For eight weeks these soils were incubated under a periodically changing temperature range of 6–30°C while frequently measuring soil microbial respiration rate (Rs; each sample about every 20 minutes). This experimental design allowed us to investigate Rs and the temperature sensitivity of Rs (Q10) along the NSTEC. Both Rs at 20°C (R20) and Q10 significantly increased (logarithmically) with increasing latitude along the NSTEC suggesting that the sensitivity of soil microbial respiration to changing temperatures is higher in forest soils from locations with lower temperature. Our findings from an incubation experiment provide support for the hypothesis that temperature sensitivity of soil microbial respiration increases with biochemical recalcitrance (C quality-temperature hypothesis) across forest soils on a large spatial scale. Furthermore, microbial properties primarily controlled the observed patterns of R20, whereas both substrate and microbial properties collectively controlled the observed patterns of Q10. These findings advance our understanding of the driving factors (microbial vs. substrate properties) of R20 and Q10 as well as the general relationships between temperature sensitivity of soil microbial respiration and environmental factors.