The rice-wheat rotation system in Jintan of Jiangsu Province was chosen to investigate the effects of rice straw returning combined with fertilizer application on soil nutrients, microbial biomass, crop yield and nitrogen use efficiency in wheat season via filed experiments. Results showed that the contents of soil available nutrients could be increased by rice straw returning combined with fertilization. The treatment of fertilization only significantly affected soil microbial biomass C at jointing stage, while rice straw incorporation notably influenced soil microbial biomass N at jointing and heading stages, and the interaction of rice straw returning and fertilization observably affected soil microbial biomass quotient at jointing stage. The nitrogen use efficiencies of fertilization and rice straw returning combined with fertilization treatments were 31% and 37%, respectively; while the nitrogen agronomic efficiency and partial factor productivity showed that the yield increase per kilogram pure nitrogen was about 6.86 kg grain. The spikelets per panicle and 1 000-grain weight of fertilization and rice straw returning combined with fertilization treatments were significantly increased. The theoretical yields of these two treatments increased by 211% and 319%, respectively, while actual yields increased by 119% and 231%, respectively. However, the actual yield of rice straw returning decreased by 21%. Overall, rice straw returning to the field combined with fertilization can ensure the supply of soil available nitrogen, facilitate the transformation of soil organic matter into microbial available form, improve the nutrient availability, promote the absorption and utilization of nitrogen by wheat, and is to the benefit of spikelets per panicle and 1 000-grain weight increases, thus ultimately elevate wheat yield.