Abstract: The alteration of rainfall pattern under the background of global climate change may affect the quantity and quality of soil dissolved organic matter (DOM). To better understand the responses of soil DOM to rainfall reduction in subtropical forests, we conducted a 6-year rainfall reduction experiment. There were three treatments: control (CK), 30% rainfall reduction (-30%), and 60% rainfall reduction (-60%). With ultraviolet-visible, infrared and three-dimensional fluorescence spectroscopy, we investigated the effects of rainfall reduction on the quantity and structure of DOM from different soil layers of a natural Castanopsis carlesii forest in subtropical China. Rainfall significantly reduced the content of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) in the 0-10 cm soil layer. Specifically, less decreases of DOC than DON were found under the -30% treatment, while more reduction of DOC than DON for the -60% treatment. Under each treatment, the contents of DOC and DON were significantly higher in the 0-10 cm soil layer than that in the 10-20 cm soil layer. Relatively more microbial metabolite with complex structures, such as aromatic humus and alkanes, contributed to soil DOM from the -30% treatment than that from the -60% treatment. Beyond water availability, microbial activity was a key factor regulating the quantity and structure of soil DOM in response to rainfall reduction.