Optimal plant density and nitrogen (N) application rate are important to achieve high maize yield. High plant density increases plant-plant competition for light and nutrients. However, too much applied N promotes excessive vegetative growth and delays maturity, resulting in low N use efficiency (NUE) and potentially environmental problems. The physiological and molecular mechanisms behind the interaction between plant density and N application rate are largely unknown. We hypothesized that simultaneously improving maize yield and NUE depends on fine regulation of nitrogen partitioning in response to the interaction between nitrogen applications and plant densities. With a 2-year field experiment, we performed phenotypic, physiological, and gene expression analyses under two plant densities (low density of 60,000 plants ha-1 and high density of 90,000 plants ha-1) and three nitrogen application levels 115 (N1), 190 (N3) and 430 (N4) kg N ha-1 in the high N-responsiveness maize cultivar ZD958. By labeling maize with 15N, 2-year results showed that both 15N uptake per plant and 15N partitioning to ear decreased under high-density conditions. Compared to N1 treatment, application of N to N3/N4 level increased maize yield by 13.9%− 43.2%, which can be explained by bigger root angle, larger leaf area and lower lodging rate under high density condition. In addition, results in 2019 experiment showed that yield, NUE, nitrogen allocation to stem and the expression of some N transporter genes were regulated by the interaction between nitrogen applications and plant densities. Compared to the N4 treatment, N3 treatment has a 55.8% reduction in N, a tendency of increase in population yield, and increased NUE by 59.6% and 63.0% in low-density and high-density plantings, respectively. Our proposed model suggested that the appropriate N supply increased NUE by either increasing N translocation from leaves to ears under low-density conditions or by increasing N allocation to ears rather than to stems under high-density conditions. Based on correlation analysis, N allocation/translocation and expression of nitrogen transporter genes could be used as biomarkers to indicate appropriate levels of N application under different planting densities, and are thereby important for the simultaneously improving ZD958 yield and NUE in intensive agriculture.