The root system plays an important role in the growth and development of cotton, and root growth is closely related to shoot growth, both of which are affected by N availability in the soil. However, it is unknown how N affects root growth and the rootshoot relationship under various N rates in the Yellow River Basin, China. Thus, the aim of this study was to assess the impacts of the application rate of N on root growth and the root-shoot relationship, to provide insight into the N regulation of root and shoot growth and N efficiency from the perspective of the root system. A field experiment conducted in 2014 and 2015 was used to determine the effects of N rates (0, 120, 240, and 480 kg ha −1) on root morphology, root distribution, the root-shoot relationship, and cotton yield. A moderate N fertilization rate (240 kg ha −1) increased root length, root surface area, and root biomass in most soil layers and significantly increased total root growth and total root biomass by more than 36.06% compared to the 0 kg ha −1 treatment. In addition, roots in the surface soil layers were more strongly affected by N fertilization than roots distributed in the deeper soil layers. Total root length, total root surface area, and root biomass in the 0-15 cm layer were significantly correlated with shoot biomass and boll biomass. In the 60-75 cm layer, total root length, total root surface area, and root length were significantly positively correlated with seed cotton yield. The application of a moderate level of N markedly increased total shoot biomass, boll biomass, and seed cotton yield. Our results show that increased shoot and boll biomasses were correlated with a significant increase in the root system especially the shallow roots in the moderate N treatment (240 kg ha −1), leading to an increase in cotton seed yield.