AbstractAtmospheric nitrogen (N) deposition rates affect soil N dynamics, influencing soil respiration (R_S) rates. However, for the Mediterranean region, the effect of changes in atmospheric N deposition on R_S are not well constrained yet. We investigated the interplay between increased N deposition and tree species composition on R_S at a Scots pine—Pyrenean oak ecotone in Central Spain, and whether the observed responses were mediated by changes on selected soil properties. Throughout 3 years, we simulated two N deposition rates—10 (medium) and 40 kg N ha⁻¹ a⁻¹ (high)—over the background deposition (control) in neighbouring stands in which tree species composition (pine or oak) shapes soil stoichiometry and microbial communities. We monitored R_S on a monthly basis during 3 years; in addition, we performed targeted measurements 24 h after the N fertilization events to assess short-term soil responses. During winter and summer, R_S did not respond to enhanced N deposition rates. In spring and autumn, higher R_S rates were observed in the medium-fertilization, but the size and duration of this effect was tree species dependent. We suggest that climate seasonality modulates the response of R_S to N availability, with tree species effects becoming relevant only when environmental conditions are adequate. R_S in fertilized plots was larger from February to May and in September under pine, while under oak a response was observed only in April, probably due to differences in native soil stoichiometry under each tree species. Overall, R_S showed high stability during 3 years of N enrichment in this Mediterranean ecotone area. However, we observed short-term soil responses after N fertilization events—loss of base cations, soil acidification and reduced microbial biomass—which emphasize the need to investigate consequences for the belowground C and N cycles if chronic N enrichment persists in the long run.