The formation of economic yield and fruit quality of Roxburgh rose (Rosa roxburghii Tratt.) depends essentially on its source-sink interaction. Thus, a pruning experiment was conducted to assess the effects of source-sink regulation on photosynthetic physiology and fruit yield of Roxburgh rose, which was of great significance to production. Cutting off vegetative branches reduced physiological fruit loss and malformed fruits but increased single fruit quality and yield. Results revealed that the stomatal characteristics, the composition of mesophyll tissue, and photosynthesis of leaves on reproductive branches were significantly affected by the ratio of the vegetative and reproductive shoots. Our data indicated that the source-sink ratio could reflect the balance between vegetative growth and reproductive growth of the tree during the whole fruit period. Fruit tree pruning had guiding significance for improving the fruit yield of Roxburgh rose.