Rainfall significantly affects soil respiration rates by altering microbial activity and organicmatter decomposition. In karst regions, it also impacts carbonate dissolution and precipitation,further influencing soil CO2 flux. Investigating the mechanism of rainfall’s impact on soil respirationis essential for accurately evaluating and predicting changes in terrestrial ecosystems. However,our understanding of the interaction between rainfall and soil respiration in the extensive karstecosystems of southwestern China remains limited. This study conducted field-based simulatedrainfall experiments to examine variations in soil respiration rates and elucidate the associated controlmechanisms through stable carbon isotope composition analysis. Simulated rainfall significantlyincreased the CO2 release via soil respiration. We observed significant differences in the δ13C valueof soil-respired CO2 before and after simulated rainfall. Following the rain, the δ13C of soil-respiredCO2 was enriched compared to that before the rain. Through isotope data analysis, we found that theincreased soil CO2 emissions were primarily driven by heterotrophic respiration, likely stimulatedvia changes in soil moisture, affecting microbial growth conditions. Furthermore, the variation insoil moisture affected carbonate dissolution and precipitation, potentially increasing the soil CO2release after rainfall. In conclusion, these findings expand our understanding of rainfall’s effects onsoil respiration in the native karst forests of southwestern China, contributing to the prediction ofcarbon cycling processes in such ecosystems. The data from this study have significant implicationsfor addressing the release of greenhouse gases in efforts to combat climate change.