Conservation tillage is a key technology in the trend towards sustainable agriculture. The main targets of conservation tillage are to reduce soil erosion and protect the soil and the environment. Conservation tillage can be complemented by the use of a range of other cultivation techniques, including no-till cultivation techniques, the use of straw stubble technology and green covering technology. There are a number of current studies reporting on the effects of different tillage methods on crop yield and soil nutrients. There have been fewer comparisons of the effects of long-term application of conservation tillage on rice yield, soil physical, chemical and biological properties and interannual variability. Through research on trends in rice yield, soil physical and chemical properties and variation of soil biological activity under different tillage methods, this study explores the advantages of long-term application of conservation tillage on rice yield and soil fertility in paddy fields. It provides theoretical support and a sound scientific basis for the rationalization and vigorous promotion of conservation tillage technology in paddy fields. We have comprehensively studied the effects of long-term application of conservation tillage on rice yield, soil fertility and biological activity. The study site is located in the southern hilly region in Jiangxi Province, China. The double-cropping experiment was conducted in a paddy field at Jiangxi Agricultural University between 2005 and 2012. The test comprised a total of four treatments: (1) conventional tillage + transplanting (CT + P), the traditional farming method; (2) conservation tillage treatment: conservation tillage + throwing (CT + T), the traditional method of plowing and throwing seedlings after flattening; (3) conservation tillage treatment: no-tillage + transplanting (NT + P), no tillage, with the herbicide applied to stubble before transplanting; (4) conservation tillage treatment: no tillage + throwing (NT + T), no tillage, with the herbicide applied to stubble before throwing. The results indicate that with conservational tillage, rice yield was significantly higher (4.5%-8.8%) than with conventional tillage. Yield components were also higher with conservation tillage than conventional tillage, but the differences in ear length and grain weight among all treatments were not significant. Implementation of conservation tillage decreased soil bulk density by 3.6%-5.6%, increased total porosity by 1.6%-17.4% and increased capillary porosity by 2.4%-16.7% when compared with conventional tillage. Significant increases in soil organic matter (2.9%-10.0%), available phosphorus (4.8%-31.6%) and available potassium (9.7%-25.7%) were also obtained under long-term conservation tillage treatments when compared with conventional tillage. The abundance of fungi in the no-tillage + planting treatments was significantly higher (51.6%) than in conventional tillage in 2005, while the abundance of fungi in the no-tillage + throwing treatments was significantly higher (54.1%) than in conventional tillage in 2008. The differences in the abundance of nitrifying bacteria among all treatments were not significant between 2005 and 2011. The abundance of nitrifying bacteria in the no-tillage + throwing and the no-tillage + planting treatments were significantly higher (126.1% and 121.1%, respectively) than conventional tillage in 2012. No significant differences were obtained for catalase or urease activity among all treatments. Invertase activity varied between 0.292 and 0.451 mg/g during the 8 years sampled. Invertase activity was significantly higher in the no-tillage + throwing treatments (72.7%-137.7%) than in conventional tillage for the years 2005-2007 and in 2012. The implementation of conservation tillage was an effective model for sustainable development of agriculture and was suitable for the southern hilly region. The no-tillage + throwing and no-tillage + planting treatments generated the most significant effects.