Foraging, resting and trampling of Milu Deer could affect soil environment of the habitat, which in turn influences soil microbial community. Differences between soil bacterial and fungal community structure in the inactive and active areas of Dafeng Milu Deer National Nature Reserve, Jiangsu Province, China were analyzed by using high-throughput sequencing technology, and soil physicochemical indexes were measured as well as investigated the effects of grazing prohibition on soil microbial community structure. The results showed that the dominant phylum for bacteria was Proteobacteria and for fungi was Ascomycota. The grazing prohibition has changed the structure of soil microbial community, increasing the relative abundance of the Proteobacteria, Actinobacteriota and Basidiomycota at the phylum level, and decreasing the relative abundance of the Chloroflexi, Firmicutes and Ascomycota. The similarity in soil microbial community diversity between the inactive areas and active areas were low. In the redundancy analysis, bacteria were more influenced by soil environmental factors than fungi, with soil pH being the most important factor affecting the soil bacterial and fungal communities. This thesis reveals the effects of grazing prohibition on the structure of soil microbial communities, provides a reference for the reserve to develop a habitat restoration plan for Milu deer.