The monitoring and research of biodiversity pattern in protected area is very important not only to understand the protected objects of protected area, its roles in protected area network and regional ecological security, but also to enhance the function of protected areas. The routine patrolling and monitoring data from 2016 to 2017 was used to assess mammal diversity of Hunan Hupingshan National Nature Reserve in this paper. Hupingshan Reserve set 25 investigating transects with average 7 km length at 8 monitoring stations. Each transect was investigated 3 times per month. During these two years, 25 mammal species were recorded in total, accounting for 26.31% mammal species of Hunan Province. We calculated the alpha diversity index of each monitoring station and beta diversity index of whole reserve, and compared the Hellinger distance of species composition among stations in different seasons. The results showed that there was significant difference among mammal diversity in different inter-areas and monitoring stations. The mammal diversity level in core zone and relatively well-protected areas was significantly higher than that in other areas. The species compositions were significant different seasonally among different monitoring stations. The abundance of ungulates was relatively high because of lacking large predators. It is necessary to improve the methods and efficiency of routine monitoring for Hupingshan Nature Reserve. The ungulate control predetermined plan should be based on the monitoring results in order to stabilize the structure and function of reserve ecosystem.