Abstract Wheat–cotton rotations largely increase crop yield and improve resources use efficiency, such as the radiation use efficiency. However, little information is available on the nitrogen (N) utilization and requirement of cotton under wheat–cotton rotations. This study was to determine the N uptake and use efficiency by evaluating the cotton ( Gossypium hirsutum L.) N use and the soil N balances, which will help to improve N resource management in wheat–cotton rotations. Field experiments were conducted during 2011/2012 and 2012/2013 growing seasons in the Yangtze River region in China. Two cotton cultivars (Siza 3, mid-late maturity with 130 days growth duration; CCRI 50, early maturity with 110 days growth duration) were planted under four cropping systems including monoculture cotton (MC), wheat/intercropped cotton (W/IC), wheat/transplanted cotton (W/TC) and wheat/direct-seeded cotton (W/DC). The N uptake and use efficiency of cotton were quantified under different cropping systems. The results showed that wheat–cotton rotations decreased the cotton N uptake through reducing the N accumulation rate and shortening the duration of fast N accumulation phase as compared to the monoculture cotton. Compared with MC, the N uptake of IC, TC and DC were decreased by 12.0%, 20.5% and 23.4% for Siza 3, respectively, and 7.3%, 10.7% and 17.6% for CCRI 50, respectively. Wheat–cotton rotations had a lower N harvest index as a consequence of the weaker sink capacity in the cotton plant caused by the delayed fruiting and boll formation. Wheat–cotton rotations used N inefficiently relative to the monoculture cotton, showing consistently lower level of the N agronomic use efficiency (NAE), N apparent recovery efficiency (NRE), N physiological efficiency (NPE) and N partial factor productivity (NPFP), particularly for DC. Relative to the mid–late maturity cultivar of Siza 3, the early maturity cultivar of CCRI 50 had higher N use efficiency in wheat–cotton rotations. An analysis of the crop N balance suggested that the high N excess in preceding wheat ( Triticum aestivum L.) in wheat–cotton rotations led to significantly higher N surpluses than the monoculture cotton. The N management for the cotton in wheat–cotton rotations should be improved by means of reducing the base fertilizer input and increasing the bloom application.