Nitrogen (N) loss is becoming one of the main limitations for sustainable agricultural production globally, particularly for crops such as cotton. To alleviate this issue, a promising strategy may be to exploit the N-saving potential of cotton plants by optimizing N application ratios at different growth stages under lower N rate and higher planting density with wide-row spacing. Two field experiments were carried out to investigate cotton yield, N use efficiency (NUE), N accumulation characteristics, and 15N uptake and distribution in response to N fertilization. First, a two-year experiment was conducted with N fertilization of either 0, 120, 240, 360 (control) or 480 kg ha−1. A three-year experiment was then carried out with 240 kg N ha−1 being applied with different ratios across three periods (squaring, flowering to peak boll, and late peak boll), i.e., 0:6:4 (N064), 1:6:3 (N163), 2:6:2 (N262, control), 3:6:1 (N361) and 4:6:0 (N460). Compared with conventional N fertilization (360 kg N ha−1), moderate fertilization at 240 kg N ha−1 resulted in steady and higher seed cotton yields of 5945 and 5603 kg ha−1 in 2017 and 2018, respectively, and improved NUE by 49.1–53.6%. Compared with conventional N262, a shift to later N application (N064) increased both lint yield and partial factor fiber productivity of nitrogen (PFFPN) by 4.4–7.7%, and accumulated 6.1–14.4% more total N (K) in reproductive organs due to higher average (Vt) and maximum (Vm) accumulation rates by 25.2–49.0% and 15.0–48.7%, respectively, while the fast N accumulation period (∆t) shortened by 15.2–24.4%. N064 partitioned 15.6% more 15N to reproductive organs. NUE was significantly positively correlated with K, t1 (the day when ∆t started), Vt, Vm, Tm (the day on which Vm occurred) and 15N accumulation in middle and upper canopy bolls, and negatively correlated with ∆t of total N accumulation in reproductive organs. Allocating N from the squaring period to the late peak boll period led to an increase in N uptake during the reproductive growth stage and greater allocation of N to cotton bolls under reduced-N cultivation and drip fertigation. This N management strategy is a potential way to improve NUE and achieve sustainable and efficient production of cotton in arid areas.
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