Abstract: In order to study changes of physical and chemical characteristics and microbial activities in soil under different tillage methods, effects of four tillage methods, rotary tillage (RT), subsoil tillage (ST), conventional tillage (CT) with corn straw returned to soil, and rotary tillage with no corn straw returned to soil (CK), on phospholipid fatty acids (PLFA) characteristics and hydrolase enzymes activities in calcareous cinnamon soil were investigated. The results showed that soil hydrolase enzymes activities, nutrient contents, microbial diversity varied greatly with the different tillage methods. Returning corn straw to soil increased the kinds, amount of soil total PLFAs, bacteria PLFAs and actonomycetes PLFAs, while decreased the fungi PLFAs, indicating that fungi was more adaptable than bacteria to an infertile environment. ST and CT resulted in higher amounts of total PLFAs, which were 74.7% and 53.3% higher than that of CK, indicating they were more beneficial to the growth of plants. They could also improve soil physical and chemical properties, increase alkphosphatase, protease and urease activities, which would provide a favorable soil condition for high and stable crop yields.