Overwintering and succession of phytoplankton community with physicochemical and biological characteristics were investigated in pilot culture system. Water and phytoplankton samples were collected twice a week from February 23 to June 28, 2016. A total of 17 overwintering taxa including cyanophyceae, chlorophyceae, bacillariophyceae were identified in the experimental group in winter (February), and these overwintering species showed a marked succession pattern along with environment changes. In the process of phytoplankton succession, a total of 56 species in 28 genera were identified in two (experimental, control) pilot culture system. In the experimental group, 52 phytoplankton species in 24 genera were identified, and the number of taxa was highest in chlorophyceae (35 species), followed by Bacillariophyceae (9 species), Cyanophyceae (5 species) and others (3 species). In the control group, 25 phytoplankton species in 14 genera were classified and these taxa consisted of 17 chlorophyceae, 3 cyanophyceae, 2 Bacillariophyceae and 3 others. The standing crops ranged from 40 to 325,450 cells mL-1 in the experimental group, and from 900 to 37,100 cells mL-1 in the control group, respectively. The dominant species were represented by Monoraphidium minutum, Microcystis aeruginosa, Rhodomonas lacustris, Ankyra judai and Chlorella vulgaris in the experimental group; and M. minutum and Coenochloris cf. pyrenoidosa in the control group. In conclusion, overwintering and succession of predominant phytoplankton species developed due to interactions of internal environmental factors (physicochemical and biological factors) in the pilot culture system.