The soil microbial community is a key indicator used to evaluate the effectiveness of land use changes in degraded ecosystems. However, there is currently limited information on the vertical variations of soil microbial communities under different cropland types in the karst region of southwest China. In this study, three different cropland types (sugarcane, mulberry, and forage grass) were selected to replace maize-soybean cultivation in a karst area of southwest China. Secondary forest was included for comparison. Soil was collected at soil depths of 0-10cm, 10-20cm, 20-30cm, and 30-50cm to conduct a phospholipid fatty acid (PLFA) analysis of the soil microbial communities. The results showed that the abundances of total PLFAs and PLFAs of bacteria, fungi, actinomycetes, and arbuscular mycorrhizal fungi were significantly higher in forage grass cultivation and secondary forest areas than those in other three cropland types. The abundance of total PLFA and its functional groups significantly decreased with increasing soil depth. Bacteria was the predominant community (> 52%) across all land use types and soil depths. A principal component analysis indicated that soil microbial PLFA composition in forage grass field and secondary forest areas differed significantly from that in mulberry, sugarcane, and maize-soybean fields across 0-50-cm soil layer. Soil organic carbon was identified as the primary factor affecting the abundance and structure of soil microbial communities in the four soil layers. These results suggest that greater improvements in soil quality and fertility could be made by replacing the maize-soybean rotation system with forage grass cultivation than by replacing it with mulberry or sugarcane cultivation in the karst region of southwest China.