At present, most lakes in the world are facing the threat of eutrophication, especially mesotrophic lakes, which are in a critical period of ecosystem transformation. However, how the internal biological composition and structure of these lakes change accordingly has not yet been explored extensively. Here, a paleolimnological evaluation of subfossil Cladocera was initiated to study ecological and hydrological environmental changes in Nanyi Lake, which is a large (203 km²) shallow freshwater lake in the lower reaches of the Yangtze River Basin, China. In this study, subfossil Cladocera data combined with several geochemical proxies from three well-dated sediment cores were obtained. Both the sedimentary subfossil Cladocera and the modern Cladocera in the water column indicated that Bosminidae was the dominant species in Nanyi Lake. We observed that the changes in the density of Bosminidae and Cladocera had strong ties to changes in the cultivated land area, which could cause the obvious alteration of the sedimentation rate around the lake. In addition, the ratio of planktonic species to littoral species (P/L ratio) in Nanyi Lake responded to the occurrence of several extreme hydrological phenomena, such as flooding corresponding to a high P/L ratio and drought corresponding to a low P/L ratio, during the past 200 years. The analysis of Cladocera assemblages and the geochemical indices of the three cores in Nanyi Lake indicated increases in trophy and water levels, especially since the modern 1970s.