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Effects of long-term fertilization and land management on the microbial communities in a Chinese Mollisol were investigated by the determination of phospholipid fatty acids (PLFAs), soil microbial biomass C or N (Cmic or Nmic) and soil respiration. Compared to CK and NPK, farmyard manure (FYM) application and fallow constantly maintained high Cmic-to-Corg ratios, high Cmic (Nmic) and PLFAs contents, and low qCO2 value. Mere chemical NPK did not improve the nutrition status but promoted Corg loss and inhibited microbial growth. PCA of PLFAs showed that the microbial community structure in arable fields (with FYM or not) and fallow were significantly different from each other. The loading values for the individual PLFA showed that fungal PLFAs were mainly affected by fallow management, while most bacterial PLFAs were affected by FYM application.