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Cyanobacteria play important roles in carbon and nutrient cycling in aquatic systems. Cyanobacteria biomass in most lakes has increased over recent decades, threatening both water quality and ecosystem management. However, due to lack of long-term monitoring, the factors that control the interannual variability and diversity of cyanobacteria are poorly known. In this study, we used genetics and geochemical record of a vertical-down sediment core to investigate the impacts of climate changes and human activities on the long-term (100-year) abundance and diversity of cyanobacteria at Lake Chenghai, southwestern China. The results show that before 1980 CE, the nutrient level of Lake Chenghai was in a generally natural state, and human impacts were relatively weak; whereas after 1980 CE the cyanobacterial biomass inferred from both qPCR and Miseq sequencing have significantly increased compared with the average of the past 100 years, suggesting that major changes in both human activities and hydrological conditions might have occurred since then. The phosphorus (P) concentration and lake level changes are thought to have significant impacts on the cyanobacterial biomass in this lake. We contend that both human activities and the decrease in lake water level (which could lead to enrichment of nutrients in the water) could have resulted in an increase in cyanobacterial biomass and total phosphorus (TP) in lake sediments. The results of this study suggest that controls on nutrient inputs and lake levels are necessary to ensure the sustainability of Lake Chenghai, especially under a continuous global warming scenario.