Abstract: In order to studying the effects of nitrogen (N) level on nitrogen use efficiency and lint yield of cotton, Several field experiments with different levels of N application were carried out in Nanjing and Anyang, standing for the ecological conditions in the middle lower reaches of Yangtze River Valley and Yellow River Valley in China, respectively. The results indicate that the dynamic developments of nitrogen use efficiency (NUE) with time during plant-growing period presented with S curve. The NUE and momentary NUE registered their highest value at the nitrogen application level of 360kg·ha-1 in Anyang and 240kg·ha-1 in Nanjing. The lint yield increased with the increasing N rate of supply, and registered its highest value at 360 kg·ha-1 and 240 kg·ha-1 for Anyang and Nanjing, respectively. The weight per boll and boll number per plant were the most important contributors to yield, and received theirs highest values with 240 kg·ha-1 and 360 kg·ha-1 N rate of application respectively.