Lake Winnipeg has been subject to intense eutrophication and invasive species such as Rainbow Smelt and Eubosmina coregoni for the last 40 years. This study demonstrated significant increases in total phosphorus, total nitrogen, chlorophyll-a, overall zooplankton abundance, and specifically Cladocera, between 1969 and the first decade of the 21st century. There were specific basin differences in the long-term changes of the Cladocera community, with the species Chydorus sphaericus and Ceriodaphnia quadrangula negatively affected by high levels of phosphorus, nitrogen and chlorophyll-a. Moreover, long-term change in the Cladocera community composition and abundance throughout the years (1969-2011) during summer was correlated with intensifying eutrophication. Additionally, weak diel vertical migration in the zooplankton community was observed for the first time in Lake Winnipeg.