Carbon (C) storage in forest soils can be enhanced through increasing organic C input and decreasing soilheterotrophic respiration (RH). The inhibitory effffect of biochar on RH has been extensively studied in agriculturalsoils, while such an effffect and the mechanisms involved remain unknown in forest soils. Here, we examine theresponse of soil physicochemical and microbial properties to biochar application and how these factors mediatethe biochar-induced change in soil RH in a subtropical bamboo plantation. Our results showed that biocharapplication signifificantly reduced RH, and markedly altered most of the studied soil properties important for RH inthe bamboo plantation. Biochar application did not affffect soil temperature and no relationship between soil RHand either soil moisture or labile organic C content was observed, excluding the possibility that biochar reducedthe RH through changing soil temperature, moisture or labile organic C content, factors commonly considered tocontrol RH. As compared to the control, biochar application signifificantly increased the aromatic C content andRubisCO enzyme activity, while decreased β-glucosidase and cellobiohydrolase (CBH) activities. In addition, thesoil RH was positively (P < 0.01) correlated with β-glucosidase and CBH activities, while negatively (P < 0.05)correlated with RubisCO enzyme activity. Further, using structural equation modelling, we revealed that bicoharreduced RH through increasing the proportion of soil recalcitrant C fraction and decreasing the β-glucosidase andCBH activities in relation to the decomposition of carbohydrates and celluloses in the soil. This is the fifirst reportthat increased soil organic C recalcitrancy and decreased activities of C-degrading enzymes are responsible forbiochar to reduce RH in the subtropical plantation, which may be key to regulating RH in subtropical plantationsthrough forest management.