<p>Lakes are expected to experience longer summer stratification and shorter winter mixing due to climate-induced warming. These changes will impact biogeochemical cycles, but how shifts in mixing might influence lake nitrogen removal via denitrification remains unconstrained. Here we used ¹⁵N-tracer assays, molecular techniques and flux measurements to establish the seasonal dynamics of denitrification in a eutrophic lake in Switzerland. We find that denitrification was disproportionately active during the winter mixed regime, potentially driven by a previously unrecognized chitinolytic–denitrifying microbial consortium. Moreover, denitrification was strongly governed by the relative availabilities of particulate organic carbon and nitrate. Leveraging these insights enabled accurate simulation of denitrification in a lake model, revealing that a worst-case climate scenario may shorten the mixing period by ~27 days and reduce denitrification by 8–13%, increasing nitrogen export to downstream ecosystems. We conclude that lake microbial denitrification, and its associated denitrifying consortium, will be weakened by climate change.</p>