Assessing the impacts of human activities on landscape fragmentation of the Northeastern China Tiger and Leopard National Park (NCTL-NP) is of great significance for balancing the conflict between the wildlife protection and urbanization, and optimizing the habitat of the tiger and leopard. In this paper, we designed three steps to evaluate the impact of human activities on landscape fragmentation of the NCTL-NP. Firstly, according to the human footprint theory, we selected five indexes, population density, land-use status, industrial and mining activities, accessibility, and nighttime lights intensity to establish a localized index system of human footprint. Then, we selected patch density (PD), edge density (ED), landscape division (DIVISION), and Shannon's diversity index (SHDI) at the landscape scale to represent the degree of landscape fragmentation. Finally, we analyzed the impact of human footprint on the landscape fragmentation and its spatial variation in the three functional zones (core reserve, the potential habitats of the tiger and leopard, and population agglomeration zone) of the NCTL-NP. The results showed that high and very high fragmented areas, accounting for 8.96% of the NCTL-NP, were mainly distributed in the population agglomeration zone and covered up to 60.54% of the zone. Overall, the factor influencing landscape fragmentation most was land-use status, followed in order by accessibility, population density and industrial and mining activities. While the nighttime light intensity was not significantly related to landscape fragmentation. These trends were found similar in the core reserve and the potential habitats of the NCTL-NP, while the impact of industrial and mining activities on landscape fragmentation in the population agglomeration zone, although still smaller than land-use status, was larger than accessibility and population density. In terms of regions of the dominant human footprint indexes, the areas where the landscape fragmentation is mainly influenced by land-use status were the largest. The areas where the landscape fragmentation was most influenced by accessibility were distributed in the surrounding areas of Chunhua Town, Daduchuan Town and Chunyang Town. The areas where the landscape fragmentation was most influenced by population density were distributed in the surrounding areas of Luozigou Town and Fuxing Town. The area where the landscape fragmentation was most influenced by industrial and mining activities was the smallest, and it was mainly distributed in the surrounding areas of Luozigou Town. We suggest that the government should take some measures to reduce human activities in areas with severely fragmented landscapes to optimize the habitat of the tiger and leopard.