Rainfall significantly affects soil respiration rates by altering microbial activity and organic matter 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 respiration is essential for accurately evaluating and predicting changes in terrestrial ecosystems. However, our understanding of the interaction between rainfall and soil respiration in the extensive karst ecosystems of southwestern China remains limited. This study conducted field-based simulated rainfall experiments to examine variations in soil respiration rates and elucidate the associated control mechanisms through stable carbon isotope composition analysis. Simulated rainfall significantly increased the CO2 release via soil respiration. We observed significant differences in the δ13C value of soil-respired CO2 before and after simulated rainfall. Following the rain, the δ13C of soil-respired CO2 was enriched compared to that before the rain. Through isotope data analysis, we found that the increased soil CO2 emissions were primarily driven by heterotrophic respiration, likely stimulated via changes in soil moisture, affecting microbial growth conditions. Furthermore, the variation in soil moisture affected carbonate dissolution and precipitation, potentially increasing the soil CO2 release after rainfall. In conclusion, these findings expand our understanding of rainfall’s effects on soil respiration in the native karst forests of southwestern China, contributing to the prediction of carbon cycling processes in such ecosystems. The data from this study have significant implications for addressing the release of greenhouse gases in efforts to combat climate change.