Abstract: The traditional cotton transplanting technique was adopted as an intercropped planting system to improve comprehensive harvest gains in limited acreage fields. However, this method was not suitable for current cotton production because of self-evident production barriers such as intensive labor requirement and overuse of nitrogen fertilizers. The new cotton planting system (field-seeding after barley harvest) without transplanting is more economical in terms of labor input, and therefore more convenient for mechanized cotton production. It is the now prospective direction of cotton production in Yangtze River Valley. To optimize the cultivation strategy of this new cotton cultivation system, the adoption of short-season cotton varieties and simplification of nitrogen application are the key techniques under consideration for the effect of the shortened growth duration of cotton after barley harvest. The objective of the study was to explore the effect of different nitrogen management schemes on yield, biomass and nitrogen use efficiency of short-season cotton variety (cv. CCRI 50) sowed after barley harvest, and to recommend the appropriate nitrogen management scheme under the cultivation system. A field experiment was conducted in 2013 and 2014 at the experimental station of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province, China. A split-plot design with three replicates was adopted where the main plot factor was nitrogen application rate and the sub-plot factor was application frequency. There were 5 levels of nitrogen application rate 0 kg(N)·hm^-2, 75 kg(N)·hm^-2, 150 kg(N)·hm^-2, 225 kg(N)·hm^-2 and 300 kg(N)·hm^-2 and 2 levels of application frequency (1 or 2 times). Biomass and yields, nitrogen use efficiency of cotton were investigated. The results showed that cotton lint yield significantly increased following an increase in nitrogen rate from 0 to 150 kg(N)·hm^-2. The cotton lint yield in 2 times fertilizer application treatment was higher than that of one time fertilizer application. The interactive effect of nitrogen rate and application frequency showed that the cotton lint yield was highest under 150 kg (N)·hm^-2 nitrogen rate and 2 times application of fertilizer condition. Biomass and nitrogen accumulation also increased with increasing nitrogen rate and application frequency, while reproductive organ distribution ratio decreased. When nitrogen rate exceeded 75 kg(N)·hm^-2, apparent nitrogen recovery efficiency (ANRE), agronomic nitrogen efficiency (ANE) and nitrogen production efficiency (NPE) decreased with increasing nitrogen application rate. ANRE and ANE in the 2 times application treatment were higher than those in the one time nitrogen application treatment, but NPE showed the opposite trend. The interactive effects of nitrogen rate and application frequency showed that under 75–150 kg(N)·hm^-2 nitrogen application condition, ANRE and ANE with 2 times nitrogen application, while NPE with one time application were higher than others treatments. Correlation analysis showed that lint yields and biomass significantly correlated with nitrogen accumulation, but did not correlate with nitrogen distribution coefficient. Nitrogen utilization rate did not correlate with cotton lint yield. As a result, the optimum nitrogen management was 150 kg(N)·hm^-2 with 2 times application for cotton under field-seeding after barley harvest.