The interaction between the soil organic carbon (SOC) sequestration and the availability of soil nitrogen (N), as well as N application, remains unknown. We evaluated the response of SOC sequestration to exogenous C input and soil N availability in a 17-year field experiment of a winter wheat monoculture system with favorable precipitation. Four experimental treatments were included: control (CK), straw returning (S), nitrogen application (N), and the combination of straw returning and nitrogen application (S + N). N application (N, S + N) increased the wheat yield when the experimental period was increased. Under an equivalent input of N (N), the average yield of straw returning (S + N) in 17 years increased by 6.5%. The significant interactions of straw returning and N application increased the SOC content and stocks in the 0-20 cm soil (p < 0.05). Compared with no N application (S), straw returning with sufficient N (S + N) not only contributed to the increment of the SOC stocks but also improved the dissolved organic carbon, easily oxidizable organic carbon, particulate organic carbon, and microbial biomass carbon by 10.2%, 49.2%, 48.3%, and 38.1%, respectively. In addition, even though it was not straw amended, N application significantly increased the SOC content and stocks by 7.1% and 6.4%, respectively. After the input of organic materials, maintaining sufficient N is beneficial to SOC sequestration. Furthermore, these findings indicate that the importance of soil N availability on crop yield and SOC sequestration should be considered when N reduction measures are implemented in agricultural practices.