Determining critical ecological protected areas at the city (county) spatial scale is crucial for coordinating regional ecological environment management, control, and governance. It is a recognized consensus in academia that human activities significantly impact natural ecosystems. Many scholars ignore this point in the research process and only use several factors to characterize human influence. Therefore, this study takes Aksu City and Wensu County, important cities and towns in southern Xinjiang, as examples, focusing on the impact of human activities on the distribution of critical ecological protection areas. First, we simulated the range, intensity, and local natural conditions of human activities in the study area using geospatial data. We created corresponding resistance surfaces (human activity resistance surface and natural landscape resistance surface). We then assign different weights to the two resistance surfaces based on several possible scenarios, resulting in different synthetic resistance surfaces. Finally, we used the Linkage Mapper plugin to identify critical ecological reserves and compared several results. The results show that: Human activities have greatly interfered with the migration and dispersal of species, affecting the distribution of critical ecological reserves. The specific performance is that in the urban center area with high intensity of human activities, the number and location of the surrounding ecological corridors change significantly with the increase in the proportion of human activity resistance and the total area remains unchanged; As part of the ecological corridors, the ecological pinch points changes with the location of the corridor, and the whole area remains unchanged; The threshold range of the restoration value of ecological barrier points is reduced. The maximum value generated by the restoration of ecological barrier points is reduced, which shows that the restoration value of ecological barrier points decreases with increased human activities.