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Rain-induced soil CO2 pulse, a rapid excitation in soil CO2 flux after rain, is ubiquitously observed in terrestrial ecosystems, yet the underlying mechanisms in tropical forests are still not clear. We conducted a rain simulation experiment to quantify rain-induced changes in soil CO2 flux and microbial community composition in a tropical forest. Soil CO2 flux rapidly increased by ~83% after rains, accompanied by increases in both bacterial (~51%) and fungal (~58%) Phospholipid Fatty Acids (PLFA) biomass. However, soil CO2 flux and microbial community in the plots without litters showed limited response to rains. Direct releases of CO2 from litter layer only accounted for ~19% increases in soil CO2 flux, suggesting that the leaching of dissolved organic carbon (DOC) from litter layer to the topsoil is the major cause of rain-induced soil CO2 pulse. In addition, rain-induced changes in soil CO2 flux and microbial PLFA biomass decreased with increasing rain sizes, but they were positively correlated with litter-leached DOC concentration rather than total DOC flux. Our findings reveal an important role of litter-leached DOC input in regulating rain-induced soil CO2 pulses and microbial community composition, and may have significant implications for CO2 losses from tropical forest soils under future rainfall changes.