The stomatal compensation point of ammonia (χs) is a key factor controlling plant-atmosphere NH3 exchange, which is dependent on the nitrogen (N) supply and varies among plant species. However, knowledge gaps remain concerning the effects of elevated atmospheric N deposition and ozone (O3) on χs for forest species, resulting in large uncertainties in the parameterizations of NH3 incorporated into atmospheric chemistry and transport models (CTMs). Here, we present leaf-scale measurements of χs for hybrid poplar clone ‘546’ (Populusdeltoides cv. 55/56 x P. deltoides cv. Imperial) growing in two N treatments (N0, no N added; N50, 50 kg N ha−1 yr−1 urea fertilizer added) and two O3 treatments (CF, charcoal-filtered air; E-O3, non-filtered air plus 40 ppb) for 105 days. Our results showed that χs was significantly reduced by E-O3 (41%) and elevated N (19%). The interaction of N and O3 was significant, and N can mitigate the negative effects of O3 on χs. Elevated O3 significantly reduced the light-saturated photosynthetic rate (Asat) and chlorophyll (Chl) content and significantly increased intercellular CO2 concentrations (Ci), but had no significant effect on stomatal conductance (gs). By contrast, elevated N did not significantly affect all measured photosynthetic parameters. Overall, χs was significantly and positively correlated with Asat, gs and Chl, whereas a significant and negative relationship was observed between χs and Ci. Our results suggest that O3-induced changes in Asat, Ci and Chl may affect χs. Our findings provide a scientific basis for optimizing parameterizations of χs in CTMs in response to environmental change factors (i.e., elevated N deposition and/or O3) in the future.