Abstract: River system connectivity is a critical element for maintaining the integrity of river ecosystems and biodiversity. This study systematically analyzed the impact of river system connectivity restoration on aquatic biodiversity, using the Danube River Basin as a representative case study. The results revealed that over two centuries of navigation engineering and hydropower development, the disruption of longitudinal connectivity in the Danube River has accelerated the loss of historical sturgeon habitats, while the degradation of lateral connectivity has led to the ecological decline of floodplains. Consequently, native sturgeon species became endangered, and overall biodiversity declined. Through the implementation of systematic restoration projects, such as the Sturgeon 2020 program, the connectivity of the river system has been progressively restored, leading to a significant improvement in aquatic biodiversity. These experiences provide a valuable reference for the ecological restoration of large rivers in China. It is recommended that, in accordance with the Changjiang River Protection Law and the Yellow River Protection Law, ecological restoration of river corridors be carried out in the Changjiang and Yellow River basins, and that technical bottlenecks in the ecological retrofitting of dams be overcome. The restoration of fish migration channels should be incorporated into river basin management planning, and river system connectivity should be enhanced step by step across different regions.