The conservation of species diversity and improvement of forest structure are essential roles of the Natural Reserve Policy and the Natural Forest Protection Program (NFPP) in China. However, the long-term effects of NFPP are still not well-defined, and a natural reserve (Liangshui) and surrounding region were surveyed as a proxy of NFPP for approaching the protection effects. Our results showed that long-term conservation significantly altered the dominant species in the herb layer (80% of species), followed by shrub (58%) and tree layers (50%); there was a 1.6-8.0-fold increase in abundance in Corylus shrubs, Acer trees and Carex grass, but a 1.3-10.0-fold abundance decrease in larch trees, Athyrium herbs and Lonicera shrubs. In contrast, tree species diversity and distribution evenness increased by 31% and 23.4% in the reserve, respectively. Forest protection in the reserve also led to the forest structural alteration with the observation of larger-sized trees and shorter herbs, but relatively sparse forests (smaller tree density). Structural equation modeling manifested that the reserve directly altered forest structure, at a coefficient of 0.854, nearly two-fold higher than its impact on diversity (0.459) and dominant species (-0.445). The most affected parameters were plant size (trees and herbs) and tree density related to forest structure, tree diversity, herb richness and evenness for diversity traits, and Oxalidaceae and Rosaceae for dominant species. This study provides basic data that can be used to evaluate the impact of the nature reserve in NE China, and these findings can be used to guide the implementation of NFPP in the long-term in the future.