A field experiment with root separation was conducted to study the effects of root interaction in maize-soybean intercropping system on the plant growth and grain yield of maize and its rhizosphere microorganisms under different nitrogen fertilization levels (0.1, 0.3, 0.5, and 0.7 g x kg(-1)). Root interaction and nitrogen fertilization had positive effects on the plant height, leaf length and width, and leaf chlorophyll content of maize. Less difference was observed in the root dry mass of maize at maturing stage between the treatments root separation and no root separation. However, as compared with root separation, no root separation under the nitrogen fertilization levels 0.1, 0.3, 0.5, and 0.7 g x kg(-1) increased the biomass per maize plant by 8.8%, 6.3%, 3.6%, and 0.7%, and the economic yield per maize plant by 17.7%, 10.0%, 8.2%, and 0.9%, respectively. No root separation increased the quantity of rhizosphere fungi and azotobacteria significantly, as compared with root separation. With increasing nitrogen fertilization level, the quantity of rhizosphere bacteria, fungi, and actinomycetes presented an increasing trend, while that of rhizosphere azotobacteria decreased after an initial increase. The root-shoot ratio of maize at maturing stage was significantly negatively correlated with the quantity of rhizosphere bacteria, fungi, and actinomycetes, but less correlated with the quantity of rhizosphere azotobacteria. It was suggested that the root interaction in maize-soybean intercropping system could improve the plant growth of maize and increase the maize yield and rhizosphere microbial quantity, but the effect would be decreased with increasing nitrogen fertilization level.