The effects of different nitrogen fertilizer treatments on soil fertility and rapeseed yield were studied to determine the optimal nitrogen fertilization for winter rapeseed in the rice-rape rotation area of the middle and lower reaches of the Yangtze River. In this study, four fertilization treatments were set up: conventional fertilization (CK), nitrogen transfer before NPK fertilizer (CR), adding borax (CRB) and 70%NPK fertilizer +30% organic fertilizer (CM). The soil pH, contents of organic matter, total nitrogen, alkali-hydrolyzed nitrogen, available phosphorus and available potassium, and the yield and agronomic traits of rapeseed were analyzed under different nitrogen fertilizations. The results showed that: ① Compared with conventional fertilization, nitrogen fertilization did not significantly affect soil pH value, organic matter and available phosphorus content, while the total nitrogen content of soil was significantly increased (1.67% ~ 7.99%, P < 0.05). CM treatment significantly increased soil alkali-hydrolyzed nitrogen content by 12.5% (P < 0.05). CR and CRB treatments significantly increased soil available potassium content by 18.5% and 11.6% (P < 0.05). ② Compared with conventional fertilization, rapeseed yields of CR, CRB and CM were increased by 26.1%, 34.8% and 26.8%, but there was no significant difference among these treatments. In addition, the optimal nitrogen application treatments had no effect on plant height, productive branch number, branch location and productive horn number per plant. However, CRB treatment significantly increased the number of seeds per rape by 22.7% (P < 0.05). Correlation analysis showed that rapeseed yield was significantly positively correlated with soil total nitrogen content (r = 0.681, P < 0.01). It was also positively correlated with plant height (r = 0.638, P < 0.01) and the number of primary productive branches (r = 0.658, P < 0.01). The results showed that optimal nitrogen application is beneficial to improving soil fertility and rapeseed growth, which is the main reason for increasing yield. Considering soil fertility and rapeseed yield, the combined application of organic and inorganic fertilizer exhibits the best effect, and would be applied in the winter rapeseed season in the rice-rape rotation area of the middle and lower reaches of the Yangtze River.