Background Anthropogenic activities have led to increased N input and changes of N transformation processes in lake systems. However, changes in phytoplankton community structure caused by phytoplankton's preference for N utilization under the increasing N input remain poorly understood. This study used nitrate isotopes (delta N-15-NO3- and delta O-18-NO3-) to investigate seasonal differences in N cycling as well as associated response of phytoplankton biomass and community composition. Results Results showed that the average delta N-15-NO3- values in spring, summer, autumn, and winter were 7.6 +/- 0.7 parts per thousand, 6.1 +/- 0.7 parts per thousand, 5.5 +/- 1.8 parts per thousand, and 7.4 +/- 1.2 parts per thousand, respectively. Accordingly, the average delta O-18-NO3- values showed the following order: winter (12.8 +/- 1.0 parts per thousand) > summer (11.5 +/- 0.9 parts per thousand) > spring (10.3 +/- 0.9 parts per thousand) > autumn (7.9 +/- 1.7 parts per thousand). The main nitrate sources in Lake Poyang were soil N, N fertilizer, and the manure and sewage in all seasons, contributing 93.8%, 3.3%, and 2.8%, respectively. Nitrification and algal-derived nitrate (NO3--N) assimilation were the main biochemical processes affecting N. In spring, the signal of nitrification was stronger, while in autumn, the signals of N assimilation by algae were more pronounced. The phytoplankton community composition varied with the seasonal changes of N concentrations and forms. The total biomass of phytoplankton in winter was the lowest of all four seasons and it was negatively correlated with NO3--N concentration (P < 0.05). Conclusions The results of this study contribute to a better understanding of the role of available forms of N in floodplain lake and provide essential support for prediction of phytoplankton growth and functions. Our work deciphers the role of phytoplankton in the lake N cycle, providing theoretical support to management of phytoplankton community to future environmental changes.