Based on the long-term fertilization experiment in paddy soil (started in 1981) in the Red Soil Institute of Jiangxi Province, China, effects of fertilization methods (CK: No fertilizer; NPK: chemical fertilizer only; and NPKM: chemical fertilizer plus organic manure) on soil microbial community structure were explored using the phospholipid fatty acid (PLFA) method and BIOLOG technique. Results show that the soil applied with NPK or with NPKM was 0.91% or 3.09%, respectively, higher than the soil of CK in total PLFAs. Principal component analysis (PCA) of PLFAs shows that fertilization promoted changes in soil microbial community structure. It increased the populations of gram-negative bacteria (G^- bacteria), fungi, actinomycetes and protozoa in Treatment NPKM and the population of gram-positive bacteria (G^＋ bacteria) only in Treatment NPK, while the ratio of fungal/bacterial was found to be higher in CK than in Treatments NPK and NPKM. The microbial community structure in CK is very similar to that in Treatment NPK. Comparison between the treatments in Average Well Color Development (AWCD) shows that Treatment NPKM improved carbon utilization capacity of its soil microbial community, and hence overall activity of the soil microorganisms, while Treatment NPK weakened activity of the soil microbes. Shannon-Wiener, Simpson and McIntosh indices show that Treatment NPKM increased soil microbial community diversity, while Treatment NPK did reversely. Correlation analysis of soil PLFAs and soil nutrients shows that soil total PLFAs was extremely significantly related to soil organic matter and total nitrogen (p<0.01), but little to soil readily available nutrients.