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Phospholipid fatty acid (PLFA) can be used as biological marker of microbial biomass and community structure. In this study PLFA analysis was used on soil microbial diversity with pyrene added The results showed that in the short term, low concentrations of pyrene had no significant effect on Bac-PLFA and Fun-PLFA, while high concentration pyrene showed inhibition effect. It also revealed that every sample, after long-term treatment, showed inhibition; meanwhile the Fun-PLFA, compared with the Bac-PLFA, was more sensitive to pyrene. It was found that the effect G-/G+-PLFA had on pyrene first increased and then decreased with the increasingly higher concentration of pyrene. The principal component analysis of PLFA showed that short-term treatment with low concentrations of pyrene did not have a significant effect on soil microbial community structure, while short-term and long-term treatment with high concentrations had a significant impact on soil microbial community structure.