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.