To evaluate the effect of increasing nitrogen (N) deposition and tropospheric ozone (O3) concentrations on N-saturated forest ecosystems, we investigated the response of Japanese red pine (textit Pinus densiflora), an N-saturation sensitive tree species, to increasing N load under elevated O3 concentrations. One-year-old seedlings of red pine were treated with three levels of N supply (0, 50 and 100 mg N L−1fresh soil volume) under two levels of atmospheric O3 concentration (< 5 and 60 ppb) for two growing seasons. Nitrogen treatment did not stimulate dry matter production of the seedlings. Growth inhibition was observed in the highest N treatment under low O3 and in the two higher N treatments under elevated O3. Irrespective of the O3 concentration, increasing N supply negatively affected root growth and mycorrhizal development in fine roots, resulting in a reduction in P and Mg uptake from the soil. Net photosynthetic rate was significantly reduced by both the highest N treatment under low O3 and the two higher N treatments under elevated O3, together with decreased N-availability to Rubisco. Nitrogen assimilated from NO3−to amino acid in the needles was not affected by the treatments. However, needle protein concentration was reduced by the highest N-treatment under low O3 and by the two higher N-treatments under elevated O3. These results suggest that elevated O3 potentially disturbs the N-availability in the form of protein including Rubisco, and may advance the negative effects of excessive N-deposition on N-sensitive plant species in N-saturated forests.