To investigate the N uptake difference features between crops in the relay strip maize-soybean intercropping system, and reveal the efficient N utilization mechanism under reduced N application condition, the ¹⁵N isotope tracer and plot location experiment combined with micro plot test assays were employed in the present study. Two field experiments were performed to analyze the biomass, N uptake, N use efficiency and soil N content of maize and soybean with 3 planting patterns, including maize monoculture (MM), soybean monoculture (SS) and maize-soybean relay strip intercropping (IMS), under 3 N application rates, including no N application (NN), reduced N application (RN: 180 kg N/hm²) and conventional N application (CN: 240 kg N/hm²). The results showed that the biomass and N uptake of maize stem and grain with IMS decreased, and ¹⁵N% abundance and ¹⁵N uptake increased, compared to monoculture system; the biomass, N uptake and ¹⁵N uptake of soybean grain and plant with IMS increased significantly; and the N fertilizer utilization efficiency, soil N contribution rate, and soil ¹⁵N% abundance of maize and soybean with IMS reduced. However, the ¹⁵N recovery rate of maize and soybean increased. Comparing N application to no N application, the total N uptake, N fertilizer utilization efficiency and soil total N content of maize and soybean with N application increased, and the soil N contribution rate reduced, both in monoculture and relay strip intercropping systems; Furthermore, the ¹⁵N% abundance, ¹⁵N uptake and the recovery rate of maize aboveground plant increased significantly, but those indices of soybean decreased remarkably. Comparing reduced N application to conventional N application, the total N uptake of aboveground plant of maize and soybean with IMS raised 13.4% and 12.4%, and N fertilizer utilization efficiency raised 213.0% and 117.5%, and the soil total N content increased 12.2% and 11.6%, but the soil N contribution rate decreased 12.0% and 11.2%.In addition, the ¹⁵N uptake and recovery rate of maize plant with IMS increased 14.4% and 52.5%, for soybean, correspondingly reduced 57.1% and 42.8%. ¹⁵N abundance of maize soil with IMS dropped 6.3%, but that of soybean improved 9.2%.There were quantitative and morphological differences of N uptake in the maize- soybean relay strip intercropping system. It is conclude that reduced N application was not only benefit to the N fertilizer efficient absorption and utilization for relay strip intercropping crops, but also achieve sustained crop yield and soil fertility.