Differences in nitrogen (N) acquisition patterns between plant species are often reflected in the natural 15N isotope ratios (δ15N) of the plant tissues, however, such differences are poorly understood for co-occurring plants in tropical and subtropical forests. To evaluate species variation in N acquisition traits, we measured leaf N concentration (%N) and δ15N in tree and understory plant species under ambient N deposition (control) and after a decade of N addition at 50 kg N ha−1 yr−1 (N-plots) in an old-growth subtropical forest in southern China. We also measured changes in leaf δ15N after one-year of 15N addition in both the control and N-plots. The results show consistent significant species variation in leaf %N in both control and N-plots, but decadal N addition did not significantly affect leaf %N. Leaf δ15N values were also significantly different among the plant species both in tree and understory layers, and both in control and N-plots, suggesting differences in N acquisition strategies such as variation in N sources and dominant forms of N uptake and dependence on mycorrhizal associations among the co-occurring plant species. Significant differences between the plant species (in both control and N-plots) in changes in leaf δ15N after 15N addition were observed only in the understory plants, indicating difference in access (or use) of deposited N among the plants. Decadal N addition had species-dependent effects on leaf δ15N, suggesting the N acquisition patterns of these plant species are differently affected by N deposition. These results suggest that co-occurring plants in N-rich and subtropical forests vary in their N acquisition traits; these differences need to be accounted for when evaluating the impact of N deposition on N cycling in these ecosystems.