To explore the seasonal variations of bacterioplankton community structure in Xuanwu Lake and identify the main factors affecting bacterioplankton community composition, the bacterial 16S rRNA terminal restriction fragment length polymorphism technology and multivariate statistical methods were applied. The results show that the Invsimpson, Shannon-Wiener and Pielou indexes were the highest in summer, and the lowest in spring. Nonmetric multidimensional scaling analysis showed that bacterioplankton community structure exhibited a seasonal pattern. Mantel test, canonical correlation analysis and variation partitioning analysis show that the most significant environmental factor on the bacterioplankton community structure was temperature, and followed factor was pH. The influence of nutrient concentration was not significant. Overall, this study will be helpful for better understanding of the bacterioplankton community composition in eutrophic lakes.