Several nitrogen (N) field experiments were carried out in Nanjing and Anyang, China, to study the dynamic characteristics of biomass accumulation and N uptake, and to define the dilution curve for critical N concentration in cotton reproductive organ over the growth period. The results show that the total biomass and N accumulation were affected significantly by the rate of N application, exhibiting a sigmoid curve over time. The beginning time of fast N accumulation was 1–5 d earlier than that of biomass accumulation. The cotton lint yield was correlated with N concentration in the reproductive organ and fluctuated with varying N concentration, indicating the existence of luxurious N consumption in the cotton reproductive organ. The N concentration increased with increasing N application rates, and decreased gradually during the growth period. The relationship between biomass and N concentration can be described with a power equation. The patterns of the N concentration dilution model were consistent at both experimental sites, but the model parameter values of a differed. The results presented in this paper indicate that a critical N concentration dilution curve for cotton reproductive organ is independent of ecological region and can be described with a power equation.