Anthropogenic disturbance to natural hydrological connectivity, both longitudinal, lateral, is threatening ecological integrity of many landscapes across the globe. Floodplains of the world's large rivers, including Yangtze River, the largest river system in China, are facing ecological crisis, such as catastrophic loss of biodiversity due to reduction in hydrological connectivity. Understanding how hydrological connectivity affects dispersal potential of organisms, especially those at lower trophic levels, among habitat patches, is critical for the management of river system aiming to restore and maintain regional and local biodiversity. We evaluated the effect of river-floodplain hydrological connectivity on the macrobenthos assemblage in West Dongting Lake, a Ramsar Wetland that has been threatened mainly by anthropogenic hydrological alternation. We sampled 54 sites along the connectivity gradient from river channels (27 sites), free-connected lakes (9 sites), isolated lakes (9 sites), and modified mudflats (9 sites). Results showed that α diversity indices, including richness, Shannon-weiner index and Margalef index, were significantly different (P < 0.05) among different habitats. Connected lakes and isolated lakes had higher richness and abundance. Whereas β diversity generally decreased along the connectivity gradients; and the modified mudflats, with the lowest connectivity, had the smallest overall β diversity. More importantly, richness replacement contributed to majority of variation in community composition in river, connected- and isolated lakes, indicating that neutral dynamics under dispersal limitation might be the main process shaping the macrobenthos communities. However, in modified mudflats, the importance of nestedness was high and comparable to that of richness replacement, reflecting a non-random process of species loss resulted from niche sorting in the less favorable environment. The generalized linear model confirmed that the key driver of the spatial turnover of macro-invertebrate community composition was hydrological connectivity. Our study demonstrated hydrological connectivity is essential for maintaining ecological integrity of floodplain ecosystems.