A field study was carried out to analyze the short-term (2 years) effect of tillage and crop rotation on microbial community structure and enzyme activities of a clay loam soil. The experimental design was a split-plot arrangement of treatments, consisting of two tillage treatments-ridge tillage (RT) and no-tillage (NT)-in combination with two crop rotation treatments-corn (Zea mays L.) monoculture and a 2-year corn-soybean (Glycine max L.) rotation. Phospholipid fatty acid (PLFA) profiles were used to assess soil microbial community structure. No-tillage resulted in significantly higher total PLFAs compared to the RT treatment, which was accompanied by higher activities of protease, beta-glucosaminidase, and beta-glucosidase. This suggests a close link between soil microbial communities and enzyme activities in response to tillage. The increase of total microbial lipid biomass in the NT soils was due to the increase in both fungal and bacterial PLFAs. Crop rotation had little effect on soil bacterial communities and enzyme activities, but it significantly influenced soil fungal communities, particularly arbuscular mycorrhizal fungi. Soils under monoculture corn had higher fungal biomass than soils under corn-soybean rotation regardless of tillage treatment.