The influence of the rice straw application on rhizosphere microbial communities in the long-term rotation system remains unclear. Two experimental sites (Jingzhou and Wuxue) with the same straw fertilization have been cultivated under the rapeseed-rice rotation mode for 7years. Eco-plate data indicated that the microbial metabolic potential at both sites was enhanced in the cores applied with rice straw. Rhizosphere microbial communities at the two fields could be well distinguished by phospholipid fatty acid data. Redundancy analysis showed that the rapeseed rhizosphere microbial communities in Jingzhou were influenced by soil moisture content, available potassium and available nitrogen, whereas those in Wuxue were influenced by soil available phosphorous, available potassium, total soil carbon and available nitrogen. Variation in soil enzyme activities could be mainly explained by available potassium in Jingzhou, and it was significantly affected by total soil carbon, available nitrogen, available potassium, available phosphorous, total soil nitrogen, moisture content and total phosphorous in Wuxue. The two rapeseed rhizosphere microbial communities are differently affected by the rice-straw application. Rhizosphere with more fungi was passively influenced by the rice-straw, while that with more Gram-negative bacteria was actively in response to the treatment.