Aboveground growth is strongly influenced by belowground processes. Crop root systems have the key role in soil nutrient acquisition, with effects on nutrient balance, and improvements in root distribution increase the potential opportunities for nutrient use. Interspecific root interactions in different cropping systems are manifested as competition or facilitation . A complementary root distribution is a prerequisite for high-yields in intercropping systems . In the present study, the coexistence period between maize and soybean was approximately 8 weeks, with sampling at the grain-filling stage of maize and soybean at approximately the V3 stage, and the results showed that the ecological niche of crop roots was separated (Figs 4 and 5; S2 Table). In the coexistence period, with the increase in N levels, the roots of maize rapidly proliferated underneath the maize plants and in the middle soil layer (20–80 cm), whereas those of soybeans were primarily distributed underneath the soybean plants. Although the soybean seedlings were weak, the seedlings helped to improve soil nutrients and facilitate maize in relay intercropping , which is in contrast to a previous study in which legume seedlings were too weak to compete or facilitate with cereal in relay intercropping . This contrast may be the result of different legumes species. The CR of soybean was stronger than that of maize, and soybean performed better under RN than under CN. In a similar study, the CR of alfalfa was stronger than that of maize, and the yield advantage was greater in maize-alfalfa intercropping than that in the corresponding monocultures . Additionally, N fertilizer input was required in long-term maize-soybean relay intercropping (Tables 1 and 2, Fig 3). During the coexistence period, the demand for N was high in the maize grain-filling stage, and an insufficient supply of N would result in yield loss .