Root morphology is an important factor determining nitrogen (N) uptake by plants, which might be affected by the extent of N application. The processes associated with root morphogenesis of spring maize in response to N application rates remain poorly understood. In this study, both field and pot experiments were conducted to explore the effect of zero-N (N0), optimized-N (N180), and high-N (N360) on root morphology, anatomical structure, and N accumulation in spring maize. N application rates affected root length and surface area, and its endogenous hormone contents. The largest difference in total root length and surface area among the three N rates was found at the silking stage: the total root length and surface increased by 51.36% and 42.58% under N180 and by 7.8% and 30.14% under N360, respectively, compared with N0, and the root/shoot ratio and root bleeding sap significantly increased under N180 and N360 compared with N0. The auxin and jasmonic acid levels of roots under N180 and N360 were higher than N0. N application rates also affected root microstructure and ultrastructure. Compared with N0, the proportions of root aerating tissue under N180 and N360 were decreased by 32.42% and 11.92% at silking. The root tip cell structure was damaged under N0, and intact under N180 and N360. Moreover, the 15N allocation proportions to root and grain under N180 and N360 were increased compared to N0. Grain yields under N180 and N360 increased by 20.44% and 16.6% compared with N0, respectively. It can be concluded that optimized-N application decreased root aerated tissue and thus improved root length and root surface area through regulating auxin and jasmonic acid levels and affected N uptake and grain yield of N-efficient spring maize variety.