Disconnection between adjacent habitat patches is one of the most notable factors contributing to the decreased biotic integrity of global ecosystems. Connectivity is especially threatened in river–floodplain ecosystems in which channel modifications have disrupted the lateral links between the main river channel and floodplain lakes. In this study, we examined the interaction between the interconnectedness of floodplain lakes and main river channels and fish assemblage descriptors. Fish assemblages in two segments of an oxbow lake, one connected to and the other isolated from the Yazoo River, Mississippi, were estimated with daytime boat electrofishing during 2007–2010. The frequency of connection for the connected segment ranged from zero to seven individual events per year (mean, ∼2). The timing of most connection events reflected regional precipitation patterns. Greater species richness, diversity, and evenness were observed in the connected segment. Additionally, the connected segment had a greater abundance of piscivores and periodic life history strategists. All fishes collected solely in the connected segment were typically riverine in nature, whereas fishes collected only in the disconnected segment were more lacustrine adapted. These results suggest that periodic connection and the associated habitat heterogeneity that it provides are important for maintaining fish species richness and diversity in large-river floodplain lakes. We suggest that maintenance or restoration of connection be an integral part of fluvial ecosystem management plans.