It is recognized that the coexistence of herbaceous species in N-depleted habitats can be facilitated by N partitioning; however, the existence of such a phenomenon for trees has not yet been demonstrated. Here, we show from both foliage and soil 15N natural abundance values and from a 12-year in situ 15N addition experiment, that black spruce (Picea mariana) and jack pine (Pinus banksiana), two widespread species of the Canadian boreal forest, take up N at different depths. While black spruce takes up N from the organic soil, jack pine acquires it deeper within the highly N-depleted mineral soil. Systematic difference in foliar 15N natural abundance between the two species across seven sites distributed throughout the eastern Canadian boreal forest shows that N spatial partitioning is a widespread phenomenon. Distinct relationships between delta15N and N concentration in leaves of both species further emphasize their difference in N acquisition strategies. This result suggests that such complementary mechanisms of N acquisition could facilitate tree species coexistence in such N-depleted habitats and could contribute to the positive biodiversity-productivity relationship recently revealed for the eastern Canadian boreal forest, where jack pine is present. It also has implications for forest management and provides new insights to interpret boreal forest regeneration following natural or anthropogenic perturbations.