Abstract Ongoing global warming is expected to augment soil respiration by increasing the microbial activity, driving self-reinforcing feedback to climate change. However, the compensatory thermal adaptation of soil microorganisms and substrate depletion may weaken the effects of rising temperature on soil respiration. To test this hypothesis, we collected soils along a large-scale forest transect in eastern China spanning a natural temperature gradient, and we incubated the soils at different temperatures with or without substrate addition. We combined the exponential thermal response function and a data-driven model to study the interaction effect of thermal adaptation and substrate availability on microbial respiration and compared our results to those from two additional continental and global independent datasets. Modeled results suggested that the effect of thermal adaptation on microbial respiration was greater in areas with higher mean annual temperatures, which is consistent with the compensatory response to warming. In addition, the effect of thermal adaptation on microbial respiration was greater under substrate addition than under substrate depletion, which was also true for the independent datasets reanalyzed using our approach. Our results indicate that thermal adaptation in warmer regions could exert a more pronounced negative impact on microbial respiration when the substrate availability is abundant. These findings improve the body of knowledge on how substrate availability influences the soil microbial community–temperature interactions, which could improve estimates of projected soil carbon losses to the atmosphere through respiration.