Abstract: Changes of microorganism number and microbial biomass in soils were investigated with different years of no-tillage under the paddy-upland rotation. Results indicated that: (1) After upland cropping and paddy cropping, the number of microorganisms in no-tillage soil was significantly lower than that in conventional tillage soil. With the increase of no-tillage years, bacteria number decreased, and then appeared an increasing trend after upland cropping. The lowest number of bacteria appeared at 5–6 years of no-tillage after upland cropping treatment. No significant differences were found in soil with different no-tillage year treatment after paddy cropping. (2) After upland cropping, the number of fungi and actinomycetes in no-tillage soil were significantly higher than that in conventional tillage soil, but were lower after paddy cropping treatment. With the increase of no-tillage years, number of fungi had a trend of decreasing and then increasing, with the lowest number in soil under 7–8 year of no-tillage treatment. The amount of actinomycete kept stable after 5–6 year o f no-tillage treatment. After paddy cropping, number of fungi had a significant negative relationship with the number of actinomycetes in the soil. (3) After upland and paddy cropping, microbial biomass C and N in no-tillage soil were significantly higher than that in conventional tillage soil, the microbial biomass C and N had positive coorelations; with the increase of no-tillage years, microbial biomass C and N decreased after upland cropping, but it showed increasing and then decreasing trend after paddy cropping.