Background <p>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 (δ¹⁵N-NO₃⁻ and δ¹⁸O-NO₃⁻) to investigate seasonal differences in N cycling as well as associated response of phytoplankton biomass and community composition.</p> Results <p>Results showed that the average δ¹⁵N-NO₃⁻ values in spring, summer, autumn, and winter were 7.6 ± 0.7‰, 6.1 ± 0.7‰, 5.5 ± 1.8‰, and 7.4 ± 1.2‰, respectively. Accordingly, the average δ¹⁸O-NO₃⁻ values showed the following order: winter (12.8 ± 1.0‰) &gt; summer (11.5 ± 0.9‰) &gt; spring (10.3 ± 0.9‰) &gt; autumn (7.9 ± 1.7‰). 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 (<i>P</i> &lt; 0.05).</p> Conclusions <p>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.</p>