Abstract: By using phospholipid fatty acid (PLFA) method, this paper studied the changes of soil microbial community structure in an alpine meadow under six years continuous enclosure and its combination with fertilization, taking grazing area as the control. Both continuous enclosure and its combination with fertilization had significant effects on the microbial flora and total PLFA in different soil layers, and the effects were greater for 0-10 cm than for 10-20 cm soil layer. The species of PLFA in different soil layers also changed significantly. Under enclosure and its combination with fertilization, the number of gramnegative bacteria in different soil layers was lower than that of the control, and the numbers of bacteria, fungi, and gram-positive bacteria and the total PLFA in 0-10 cm soil layer were also lower, but the number of actinomycetes was higher than that of the control. In 10-20 cm soil layer, the number of grampositive bacteria had no significant differences among different treatments, whereas the numbers of bacteria, fungi, and actinomycetes and the total PLFA were notably higher under enclosure but decreased markedly after fertilization. As compared with that of the control, the ratio of bacteria and fungi in different soil layers under enclosure and its combination with fertilization increased, the ratios of saturated fatty acid to monounsaturated fatty acid (SAT/MONO) and of grampositive bacteria to gramnegative bacteria (G⁺/G^-) under enclosure were all lower, but those after fertilization were in adverse. It was suggested that continuous enclosure and its combination with fertilization led to the decline of soil microbial diversity and activity and soil ecosystem stability.