The response of plant CO2 diffusion conductances (mesophyll and stomatal conductances, gm and gsc) to soil drought has been widely studied, but few studies have investigated the effects of soil nitrogen addition levels on gm and gsc. In this study, we investigated the responses of gm and gsc of Manchurian ash and Mongolian oak to four soil nitrogen addition levels (control, low nitrogen, medium nitrogen and high nitrogen) and the changes in leaf anatomy and associated enzyme activities (aquaporin (AQP) and carbonic anhydrase (CA)). Both gm and gsc increased with the soil nitrogen addition levels for both species, but then decreased under the high nitrogen addition level, which primarily resulted from the enlargements in leaf and mesophyll cell thicknesses, mesophyll surface area exposed to intercellular space per unit leaf area and stomatal opening status with soil nitrogen addition. Additionally, the improvements in leaf N content and AQP and CA activities also significantly promoted gm and gsc increases. The addition of moderate levels of soil nitrogen had notably positive effects on CO2 diffusion conductance in leaf anatomy and physiology in Manchurian ash and Mongolian oak, but these positive effects were weakened with the addition of high levels of soil nitrogen.