The microbial conversion of organic nitrogen (N) to plant available forms is a critical determinant of plant growth and carbon sequestration in forests worldwide. In temperate zones, microbial activity is coupled to variations in temperature, yet at the ecosystem level, microbial N mineralization seems to play a minor role in determining patterns of N loss. Rather, N losses often appear to vary with seasonality in hydrology and plant demand, while exports over longer periods are thought to be associated with increasing rates of anthropogenic N deposition. We analyzed long-term (21–32 years) time series of climate and stream and atmospheric chemistry from two temperate deciduous forest watersheds in the southeastern USA to understand the sensitivity of internal forest N cycles to climate variation and atmospheric deposition. We evaluated the time series with a simple analytical model that incorporates key biotic constraints and mechanisms of N limitation and cycling in plant–soil systems. Through maximum likelihood analysis, we derive biologically realistic estimates of N mineralization and its temperature sensitivity (Q10). We find that seasonality and long-term trends in stream nitrate (NO3) concentrations can in large part be explained by the dynamics of internal biological cycling responding to climate rather than external forcing from atmospheric chemistry. In particular, our model analysis suggests that much of the variation in N cycling in these forests results from the response of microbial activity to temperature, causing NO3 losses to peak in the growing season and to accelerate with recent warming. Extrapolation of current trends in temperature and N deposition suggests that the upturn in temperature may increase future N export by greater than threefold more than from increasing deposition, revealing a potential direct effect of anthropogenic warming on terrestrial N cycles.