In recent decades, the global intensification of human activities had profound impacts on regional ecosystem and posed severe threats to regional sustainable development. Scientific measurement of the relationship between human activities and habitat quality can provide solid basis for the formulation of effective ecosystem protection policies. Shennongjia Forest Region, as the first heritage site in China to be awarded the three lists of "UNESCO World Natural Heritage", "World Geopark", and "Man and Biosphere Nature Reserve", which is of great ecological protection value. Due to the fact that Shennongjia Forest Region is playing an indispensable ecological barrier in China and even in the world, exploring the spatial relationship between human activities and habitat quality in Shennongjia Forest Region as well as the extent of human activities' influence on habitat quality has momentous reference and guiding significance for the coordinated development of ecosystem conservation and economic development. This study measured the spatiotemporal distribution patterns of habitat quality and human footprint with the multi-source data using InVEST model in Shennongjia Forest Region in 1995, 2000, 2005, 2010, and 2015. The spatial relationship between habitat quality and human footprint were analyzed using bivariate spatial autocorrelation and geographically weighted regression models. The results showed that: (1) the habitat quality level of Shennongjia Forest Region remained basically stable from 1995 to 2015, and over 60% of the areas were in the category of high habitat quality and higher habitat quality. (2) From 1995 to 2015, the human footprint index in Shennongjia Forest Region presented the spatial distribution patterns of low in the west, high in the northeast and the development trend of "polarization". (3) From 1995 to 2015, there was a significantly spatial dependence between human activities and habitat quality in Shennongjia Forest Region, and there was a significantly negative spatial correlation between them. In the middle of the forest region, human activities had an increasingly significant negative effect on habitat quality and human activities would lead to the deterioration of habitat quality. The results showed that the spatial dependence effect between human activities and habitat quality should be fully considered in the future ecosystem protection policy formulation, and the protection scope should be delimited scientifically and rationally to improve the implementation effect of ecological protection planning. It is recommended that future research can integrate subjective and objective methods to achieve a scientifically quantified human footprint index, and various grid scales can be utilized for comparative studies of the research area, so as to discover the associations between different variables at multiple scales and scientifically balance the relationship between human activities and ecosystem conservation.