Abstract: In order to explore the key environmental factors affecting phytoplankton community characteristics in spring and autumn in the Dongting Lake, a systematic survey of water environmental parameters and phytoplankton was conducted at 30 sampling sites of the Dongting Lake in April and October of 2018. The results showed a total of 157 species of phytoplankton belonging to 7 phyla and 51 genera were identified, and species number in April (105 species) and October (120 species) accounted for 66.9% and 76.4% of the total, respectively. The dominant groups in two surveys were all Bacillariophyta, Chlorophyta and Cyanophyta. Besides, community structure gradually changed from Bacillariophyta-dominated type to Bacillariophyta-Chlorophyta-dominated type from April to October. The average densities of phytoplankton in April and October were 19.0×104 cells/L and 18.7×104 cells/L, respectively, which indicated that abundance in two seasons did not vary significantly. The average biomass increased from 0.67 mg/L in April to 0.83 mg/L in October. Comparing the Shannon-Wiener diversity (H), Marglef diversity (d), Pielou evenness index (J) and Beta diversity in two seasons, it was found that the difference of phytoplankton community among sampling areas of the Dongting Lake in spring was larger than that in autumn, while biodiversity and richness were lower than those in autumn. The redundancy analysis (RDA) showed that pH, water velocity, conductivity and transparency were the key environmental factors affecting the phytoplankton community characteristics in April. Ammonium, water temperature, transparency and total nitrogen were the key environmental factors affecting the phytoplankton community characteristics in October. Based on previous studies in the river-connected and isolated lakes, it was found that the biodiversity of the isolated lakes were far lower than river-connected lakes, while the phytoplankton abundance and biomass of the isolated lakes were significantly higher than river-connected lakes, which was ascribed to the loss of flowing water habits and the decrease of the habitat heterogeneity in isolated lakes. It’s demonstrated that hydrological and hydrodynamic conditions are the primary factors determining phytoplankton community differences among river-connected and isolated lakes, and water physico-chemical factors (i.e., transparency, suspended solids and nutrients) are the secondary influencing factors. The results of this study can provide a scientific basis for ecological conservation of the river-connected lakes such as the Dongting Lake.