Abstract: 【Objectives】 The Yellow River Valley cotton area is one of the three largest cotton areas in China. Nitrogen fertilizer management is of vital importance in cotton production. Both deficient and excess N application in cotton crop negatively affects the lint yield and fiber quality. In this paper, a 3-years fixed plot experiment was conducted to study the effect of N application rate on the physiological characteristics of cotton functional leaves at different growth stages of cotton, and the N use efficiency and seed yield of cotton, proposing a suitable N application rate for cotton production in medium fertility field in the North China Plain Subregion. 【Methods】 A field experiment with completely randomized design and 4 replications was conducted from 2011 to 2013 on the farm of Institute of Cotton Research of Chinese Academy of Agricultural Sciences in Anyang of Henan, where the soil type is fluvo-aquic loam soil and cotton was continuously grown with transgenic Bt+CpTI cotton cultivar of CCRI(China Cotton Research Institute) 79. The effects of N application rates on leaf area index(LAI) of cotton population at different growth stages, the contents of chlorophyll, the activities of glutamine synthetase(GS) and methane dicarboxylic aldehyde(MDA) in functional leaves of cotton at different growth stages, net photosynthetic rate of functional leaves of cotton at initial flowering stage in 2013 were measured. The effects of N application rates on internal N use efficiency(iNUE,is defined as lint yield per unit tissue N), agronomic N use efficiency(aNUE, is defined as the increase in seed cotton yield per unit of fertilizer N applied), physiological N use efficiency(pNUE,is defined as the increase in seed cotton yield per unit of increased N uptake), N recovery efficiency(NRE,is defined as the increase in difference of total N uptake of N-fertilized plot and total N uptake of zero-N plot per unit of fertilizer N applied) of cotton and seed cotton yield in 2012 and 2013 were studied. 【Results】 The LAI, the chlorophyll contents and the activities of GS in functional leaves of cotton at different growth stages and the net photosynthetic rates of functional leaves of cotton at initial flowering stage were increased with the increased N fertilization rate, while the MDA contents decreased. The LAI of cotton population at peak bolling stage with the treatments of N 270 and 360 kg/ha were more appropriate than those with other N treatments, so the corresponding physiological activities in the two treatments were much higher and they provided better material insurance for high yield of cotton than other treatments. A quadratic correlation existed between the N application rates, the total N accumulation of cotton population and the seed yield. aNUE and iNUE of cotton were remarkably decreased with the increased N rates. NRE and pNUE of cotton reached the highest values at the treatment of N 270 kg/ha and N 180 kg/ha, respectively. The average seed yield of cotton of 3 years experiment reaches the highest level at the treatment of N 360 kg/ha, which was not significantly different compared with treatments of N 180, 270, 450 kg/ha, but significantly higher than the treatment of N 0, 90 kg/ha. The fertilizer effect equation between seed cotton yield and nitrogen application rate under intermediate fertility conditions in North China Plain is Y=3143.8036+4.2057N-0.006220N2(R2=0.9805, P=0.002717). The maximum and profitable cotton yield will be achieved at nitrogen application rate of 338.1 kg/ha and 299.7 kg/ha, respectively. 【Conclusions】In the medium fertility field of theNorth China Plain Subregion of Yellow River Cotton Area, the nitrogen fertilizer efficiencies of cotton will start to decline when the nitrogen application rate is more than 270 kg/ha. The recommended economical nitrogen application rate is 299.7 kg/ha for cotton in the experimental area.