Aims <p>Soil nitrogen supply capacity is primarily regulated by the transformations of organic nitrogen, which are microbial mediated processes in soils. This study aimed to investigate soil nitrogen cycling functional genes and their roles in soil nitrogen supply capacity in rice-rapeseed (RR) and rice–wheat (RW) rotation.</p> Methods <p>Soil samples were collected from a 7-year field experiment, and the abundance of soil nitrogen cycling functional genes was analyzed through metagenomic analysis. Soil inorganic nitrogen (SIN) and potentially mineralizable nitrogen (PMN) determined by anaerobic incubation were used as indicators of soil nitrogen supply capacity.</p> Results <p>Soil nitrogen supply capacity and the particulate organic matter content were higher in RR rotation than in RW rotation. The abundances of nitrogen cycling functional genes, including <i>amiABC</i>, <i>gdh</i>, <i>nxrA</i>, <i>nxrB</i>, <i>narG</i> and <i>norB</i>, were also greater in RR rotation than in RW rotation. These functional genes of nitrogen cycling involved in organic nitrogen metabolism, nitrification and denitrification processes, the abundances of these genes were significantly positively correlated with soil nitrogen supply capacity (<i>P</i> &lt; 0.05). Both the abundances of soil nitrogen cycling functional genes and the contents of soil organic carbon and total nitrogen jointly contributed to soil nitrogen supply capacity. However, the contribution of nitrogen cycling functional genes (0.34) was less than that of soil organic carbon and total nitrogen (0.67).</p> Conclusion <p>Both the advantage in the substrate quantity and availability and the subsequent advantage in the abundances of nitrogen cycling functional genes of RR rotation over RW rotation contributed jointly to greater soil nitrogen supply capacity in upland crop season in the former than the latter rotation.</p>