Abstract: The aim of this study was to investigate the effect of nitrogen and phosphorus application on maize in a maize-soybean relay intercropping system. A pot experiment was conducted in 2013 and 2014, and interspecies interactions, dry matter production and accumulation, and grain yield were assessed. The experiment consisted of four nitrogen levels [N₀ (0), N₁₂₀ (120 kg/ha), N₁₈₀ (180 kg/ha), N₂₄₀ (240 kg/ha)] and four phosphorus levels [P₀ (0), P₃₅ (35 kg/ha), P₇₀ (70 kg/ha), P₁₀₅ (105 kg/ha)]. Furthermore, to evaluate the root interaction between maize and soybean, the two plants were grown with or without a root barrier between them. The main findings were as follows: 1) The interaction between N and P significantly affected the inter-specific interaction of maize and soybean in the relay intercropping system, reduced inter-specific competition between maize and soybean, increased the land equivalent ratio, increased the aggressiveness and nutrition competition ratio, and increased the uptake of N and P. The interaction between N and P significantly increased the yield and resource utilization (land equivalent ratio>1). The interactive effect of N and P was more prominent than the effect of N or P alone in the intercropping system. 2) The effects of the N×P interaction on dry matter accumulation in each growing period of maize increased with increasing N and P application rates up to N₁₈₀ (180 kg/ha) and P₇₀ (70 kg/ha), and then decreased when N and P application rates were N₂₄₀ (240 kg/ha) and P₁₀₅ (105 kg/ha). The treatment of N₁₈₀ (180 kg/ha) and P₇₀ (70 kg/ha) significantly decreased maize dry matter accumulation before maturity, but promoted dry matter accumulation and translocation to the grain, and improved the harvest index of maize. 3) Inter-specific interactions and interactive effects of N and P were significant, and increased the yield by increasing the number of kernels per ear and the number of ears per maize plant. Among all the N and P fertilizer levels, N₁₈₀ (180 kg/ha) and P₇₀ (70 kg/ha) considerably improved the maize yield from 1.65% to 20.43% in the relay intercropping system, compared with sole cropping. On the basis of these results, we recommend nitrogen and phosphorus applications at the rates of N₁₈₀ (180 kg/ha) and P₇₀ (70 kg/ha) to substantially improve ear traits, increase dry matter translocation to the grain at maturity, and achieve the maximum yield of maize in a maize-soybean relay intercropping system.