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Corn-soybean rotation is a cropping pattern to optimize crop structure and improve resource use efficiency, and nitrogen (N) fertilizer application is an indispensable tool to increase corn yields. However, the effects of N fertilizer application levels on corn yield and soil N storage under corn-soybean rotation have not been systematically studied. The experimental located in the central part of the Songnen Plain, a split-zone experimental design was used with two planting patterns of continuous corn (CC) and corn-soybean rotations (RC) in the main zone and three N application rates of 0, 180, and 360 kg hm-2 of urea in the secondary zone. The research has shown that RC treatments can enhance plant growth and increase corn yield by 4.76% to 79.92% compared to CC treatments. The amount of N fertilizer applied has a negative correlation with yield increase range, and N application above 180 kg hm-2 has a significantly lower effect on corn yield increase. Therefore, a reduction in N fertilizer application may be appropriate. RC increased soil N storage by improving soil N-transforming enzyme activity, improving soil N content and the proportion of soil organic N fractions. Additionally, it can improve plant N use efficiency by 1.4%-5.6%. Soybeans grown in corn-soybean rotations systems have the potential to replace more than 180 kg hm-2 of urea application. Corn-soybean rotation with low N inputs is an efficient and sustainable agricultural strategy.