Our results revealed a positive interspecific facilitation between maize and soybean in the maize-soybean relay strip intercropping system. The separation of the root ecological niche contributed to interspecific facilitation. The selection of the component crop for relay strip intercropping should consider the interspecific complementary characteristics in the case of grain yield loss. For a long-term maize-soybean relay strip intercropping system, N fertilizer input is required. The total grain yield of the maize-soybean relay intercropping system increased under RN compared with that under CN. Furthermore, the nitrogen use efficiency in RN increased notably compared with that in CN in long-term maize-soybean relay strip intercropping.