Organic nitrogen (N) is the most important N component of soil organic matter. However, knowledge on how tree species with different successional stages affect organic N transformations in soils remains limited. To address this issue, we sampled mineral soils (0–10 cm) under monocultures composed of tree species from different successional stages, including early (black alder and silver birch), early to mid (sycamore and European ash), and late (sweet chestnut, pedunculate oak and European beech), and measured the potential protease activity, the microbial uptake and respiration of 14C-labeled organic N (l-alanine and l-trialanine), and the mineralization of l-alanine N. The activities of alanine aminopeptidase and leucine aminopeptidase (153.8–341.9 and 91.6–147.9 nmol/g/h, respectively), the half-life of the uptake of alanine and trialanine (26.7–39.6 and 60.8–78.6 min, respectively), the half-life of the mineralization of alanine and trialanine (1.98–2.45 and 2.98–4.13 h, respectively) by soil microbes were altered by tree species from different successional stages, systematically changing the transformation chain of soil organic N. The turnover rates of soil organic N under trees of early to late successional stage appeared to decrease and the half-life appeared to increase significantly. The C:N ratio of soil microbial biomass was positively related to the half-life of 14C-labeled alanine and trialanine mineralization, and was negatively related to the carbon (C) use efficiency of alanine, suggesting that microbial demand for C could partially drive the assimilation of soil organic N. These results suggest that the successional stage of tree species play an important role in regulating the turnover rates of soil organic N. An improved understanding of how tree species from different successional stages influence microbial function and soil organic N cycling is beneficial to future afforestation and forest management, alleviating the impacts of global change on the ecosystem.