Marco Polo sheep is a representative species in Pamir Plateau. Habitat suitability assessment is of great significance for the protection and management of the species. In this study, three groups of parameters including biological, non-biological factors, and the human interference were considered comprehensively. With help of ArcGIS, the habitat suitability index model was used to compare and analyze the potential and valid habitat areas and habitat loss rates at different scales, as well as the changes of habitat suitability in different seasons. The results showed that the potential suitable habitat areas within the reserve was 2127.19 km² in summer and 1915.70 km² in winter, respectively. The potential suitable habitat areas within the actual distribution area in the northwest area of the reserve was 1095.48 km² in summer and 1072.82 km² in winter. Therefore, the suitable habitats for the Marco Polo sheep were mainly distributed within the actual distribution area. With the human interference, the suitable habitat loss rates within the reserve were 18.43% in summer and 17.78% in winter, 33.65% in summer and 29.73% in winter within the actual distribution area. The results showed that human activities have a greater impact on the actual distribution area, so we should focus on protecting it. In conclusion, the key impact factors were the National Road G314 and grazing activities. Four suggestions and ideas are proposed for the habitat protection of the Marco Polo sheep and the management and planning of the reserve.