With continued anthropogenic inputs of nitrogen (N) into the environment, non-point source N pollutants produced in winter cannot be ignored. As the water-soil interface zones, riparian wetlands play important roles in intercepting and buffering N pollutants. However, winter has the antagonistic effect on the N removal. Substrate improvement has been suggested as a strategy to optimize wetland performance and there remain many uncertainties about the inner mechanism. This study explores the effects of substrate improvement on N removal in winter and rhizospheric crosstalk between reed (Phragmites australis) and microbes in subtropical riparian reed wetlands. The rates of wetland N removal in winter, root metabolite profiles, and rhizosphere soil microbial community compositions were determined following the addition of different substrates (gravel, gravel + biochar, ceramsite + biochar, and modified ceramsite + biochar) to natural riparian soil. The results showed that the addition of different substrates to initial soil enhanced N removal from the microcosms in winter. Gravel addition increased NH4+-N removal by 8.3% (P < 0.05). Gravel + biochar addition increased both TN and NH4+-N removals by 8.9% (P < 0.05). The root metabolite characteristics and microbial community compositions showed some variations under different substrate additions compared to the initial soil. The three treatments involving biochar addition decreased lipid metabolites and enhanced the contents and variety of carbon sources in rhizosphere soil, while modified ceramsite + biochar addition treatment had a greater impact on the microbial community structure. There was evidence for a complex crosstalk between plants and microbes in the rhizosphere, and some rhizosphere metabolites were seen to be significantly correlated with the bacterial composition of the rhizospheric microbial community. These results highlighted the importance of rhizospheric crosstalk in regulating winter N removal in riparian reed wetland, provided a scientific reference for the protection and restoration of riparian reed areas and the prevention and control of non-point source pollution.