Researching the spatial accessibility of national nature reserve would offer theoretical support for the construction and comprehensive development of regional national nature reserves. The spatial accessibility of 1 624 national nature reserves in the Yangtze River Economic Belt was estimated by using ArcGIS spatial analysis method, and the influencing factors of spatial accessibility were investigated with GWR (Geographically Weighted Regression) model. Results revealed excellent distance accessibility and time accessibility of the national nature reserves in the Yangtze River Economic Belt . A percentage of 90.14% of the national nature reserves locates within 100 km from prefectural centers, with an average time of accessibility only 24.23 minutes. The accessibility on county level display a strong agglomeration pattern, and a spatial pattern high in the east and low in the west with the line of “Chengdu-Kunming” as the dividing line. The altitude elevation of the county units is negatively correlated with the accessibility of national nature reserves. The abundance of the reserves and the density of road network are positively correlated with the accessibility. The density of road network has the greatest impact on accessibility, followed by the abundance of protected land and the elevation. The per capita GDP of county has no significant impact on accessibility. In addition, the accessibility of national nature reserves in middle and south Guizhou, southeast Yunnan, south Hunan and Anhui, as well as the coastal areas of northern Jiangsu Province is slightly sensitive to altitude elevation; the accessibility in the upper reaches of Yangtze River Economic belt is highly sensitive to road network density; the accessibility of natural reserves to the west of Chengdu-Kunming line is much more sensitive than the east.