Plant population density (PPD) and nitrogen (N) application rate (NAR) are two controllable factors in cotton production. We conducted field experiments to investigate the effects of PPD, NAR and their interaction (PPD × NAR) on yield, N uptake and N use efficiency (NUE) of cotton using a split-plot design in the North China Plain during 2013 and 2014. The main plots were PPDs (plants m−2) of 3.00 (low), 5.25 (medium) and 7.50 (high) and the subplots were NARs of 0 (N-free), 112.5 (low), 225.0 (moderate) and 337.5 (high). During both 2013 and 2014, biological yield and N uptake of cotton increased significantly, but harvesting index decreased significantly with NAR and PPD increasing. With NAR increasing, internal nitrogen use efficiency(NUE) decreased significantly under three PPDs and agronomical NUE, physiologilal NUE, nitrogen recovery efficiency(NRE) and partial factor productivity from applied nitrogen (PFPN) also decreased significantly under high PPD between two years. Lint yield increment varied during different PPDs and years, but NAR enhancement showed less function under higher PPD than lower PPD in general. Taken together, moderate NAR under medium PPD combined higher lint yield with higher agronomic NUE, physiological NUE, and NRE, while low NAR with high PPD would achieve a comparable yield with superior NRE and PFPN and high NAR under high PPD and medium PPD produced higher biological yield but lower harvest index, lint yield and NUE compared to moderate NAR with medium PPD. Our overall results indicated that, in this region, increasing PPD and decreasing NAR properly would enhance both lint yield and NUE of cotton.