An incubation test was conducted with mollisol applied with recommended amount of acetochlor under the conditions of sterilization, microbial inhibitor addition, and non-sterilization. During incubation, the residual amount of acetochlor and the dynamics of soil phospholipid fatty acids (PLFAs) were determined to study the relative contribution of soil microbes on the degradation of applied acetochlor, and the effects of acetochlor on the soil microbial community structure. The results showed that acetochlor was easy to be degraded by soil microbes, and bacteria contributed more than fungi. After applying acetochlor, the contents of various PLFAs changed evidently, and the soil microbial biomass indicated by C14:0, C16:0 and C18:0 was decreased. The bacterial PLFAs decreased significantly at the beginning of the incubation, but had less difference with CK (no acetochlor application) later, suggesting that bacterial activity was restored along with the degradation of acetochlor. The content of fungal PLFAs in the soil samples applied with acetochlor was lower than that of CK, implying that the inhibition of the herbicide to fungi was chronic and irreversible.