Abstract: The influence of different cropping systems on enzyme activities and fungal communities in representative yellow limestone soils in north Guizhou was studied over a period of 10 years in a field experiment. There were 4 cropping systems including flue-cured tobacco (Nicotiana tabacum)-wheat (Triticum aestivum) without rotation (T-W), flue-cured tobacco-canola (Brassica napus) without rotation (T-C), flue-cured tobacco-wheat-maize (Zea mays) rotation (T-W-M) and flue-cured tobacco-canola-maize rotation (T-C-M). Organic matter, microbial carbon and nitrogen, and dehydrogenase activity were increased significantly in the soil of the rotation systems, with resulting benefits to soil fertility and crop productivity. Rotation also greatly increased the number of 18S rDNA sequence, fungal phylotypes (operational taxonomic units), and the diversity index of the fungal community and reduced dominance index and the sum abundance of the top 20 predominant fungal phylotypes which suggests that rotation improved soil ecosystems. Higher diversity of fungi in soil ecosystems should prevent over reproduction of any single fungal taxon and so inhibit disease spread on a large scale. Conversely, compared with rotation, there were fewer fungal populations and a greater tendency for predominant phylotypes in soils continuously cropped under one system, which could increase the incidence of crop disease. Taxonomically, 75% of soil fungi could be assigned to Ascomycota and eight of 20 predominant fungi were commonly observed in all studied soils. Fungi in soil in the T-C system could be observed in the soils of the other three cropping systems after 10 years of the respective cropping regimes which suggests presence of those fungi is more influenced by soil environment than cropping system.