The presence of pico-sized cyanobacteria (genus Synechococcus) in hypolimnetic waters has been reported, and investigators have suggested that Synechococcus contribute more to ecological processes in the hypolimnion than previously hypothesized; however, the ecological role of Synechococcus in food webs and/or matter cycling in the hypolimnion remains unknown. To address this issue, we assessed protistan grazing and the virus-mediated mortality of Synechococcus in the oxygenated hypolimnion of a large freshwater lake (Lake Biwa, Japan) during the stratification period. In addition, we compared the carbon flux through mortality of Synechococcus to that of heterotrophic bacteria to evaluate the role of Synechococcus in ecological processes within a hypolimnetic ecosystem. Our results suggest that the biomass of Synechococcus and heterotrophic bacteria in the hypolimnion was removed primarily by protistan grazing. The abundance of Synechococcus was highest in August, when the average Synechococcus: bacteria carbon biomass and daily grazing loss ratios were 10.8% and 11.0%, respectively. Thus, the Synechococcus biomass is likely an important seasonal component of the carbon flux in the hypolimnetic microbial loop. Our results provide the first data on carbon flux through the mortality of both Synechococcus and bacteria in a hypolimnetic ecosystem.