Abstract: Effects of nitrogen application strategies which contained five different nitrogen application rates: 0, 90, 180, 270 and 360 kg N/ha and three different assigning proportions (basic N∶jointing N∶huge bellbottom N): 5∶0∶5, 3∶2∶5 and 5∶2∶3 on carbon and nitrogen metabolism of maize in wheat/maize/soybean relay intercropping system were studied by two factors randomized trials using Chuandan 418 material through two years’ field experiment. Wheat/maize/soybean relay intercropping system which was a new planting pattern could increase grain yields greatly because of soybean nitrogen fixation, inter-specific nitrogen competition and facilitation and nitrogen transfer. The results showed that, the accumulation of total sugar and nitrogen of leaves and stem/sheath and also the activity of nitrate reductase, glutamine synthetase and sucrose phosphate synthase of ear leaves which were the key enzymes to the carbon and nitrogen metabolism increased with the increasing of nitrogen fertilizer application when the amount of nitrogen fertilizer was under 270 kg N/ha, however, the results were opposite while the nitrogen levels were beyond 270 kg N/ha. For two years, the amount of nitrogen fertilizer which was 270 kg N/ha had the optimal performance, and then came 180 kg N/ha, the results of nitrogen treatment which were 180 and 270 kg N/ha were significant or quite significant to the compared group. The average decreasing rate of C/N in leaves were 26%, 25%, 21%, 23%, respectively, but in stem/sheath, it remained a relatively low level when the amount of nitrogen fertilizer was 270 kg N/ha, leaves whose nitrogen fertilizer was 270 kg N/ha still had an efficient synthetic ability in milking stage, and also remained a relatively low level of C/N in stem/sheath. Different assigning proportions had different effects on carbon and nitrogen metabolism, that was, 3∶2∶5>5∶0∶5>5∶2∶3, it was obvious that the postponing of nitrogen application could improve the matter production, and also increase the activity of nitrate reductase, glutamine synthetase and sucrose phosphate synthase of ear leaves, it was significant compared to antedisplacement of nitrogen application. As a result, nitrogen strategies in combination with wheat/maize/soybean relay intercropping system can obviously improve the carbon and nitrogen metabolism of maize, and then, realize the sustainable crop production. In combination with matter production and the performances of the key enzymes, the best nitrogen application strategy was 270 kg N/ha and 3∶2∶5.