As global temperatures continue to rise, a key uncertainty of terrestrial carbon (C)–climate feedback is the rate of C loss upon abrupt permafrost thaw. This type of thawing—termed thermokarst—may in turn accelerate or dampen the response of microbial degradation of soil organic matter and carbon dioxide (CO2) release to climate warming. However, such impacts have not yet been explored in experimental studies. Here, by experimentally warming three thermo-erosion gullies in an upland thermokarst site combined with incubating soils from five additional thermokarst-impacted sites on the Tibetan Plateau, we investigate how warming responses of soil CO2 release would change upon upland thermokarst formation. Our results show that warming-induced increase in soil CO2 release is ~5.5 times higher in thermokarst features than the adjacent non-thermokarst landforms. This larger warming response is associated with the lower substrate quality and higher abundance of microbial functional genes for recalcitrant C degradation in thermokarst-affected soils. Taken together, our study provides experimental evidence that warming-associated soil CO2 loss becomes stronger upon abrupt permafrost thaw, which could exacerbate the positive soil C–climate feedback in permafrost-affected regions.