Forest ecosystems play a vital role in global carbon (C) sequestration, which is intricately linked to root nitrogen (N) uptake. However, the strategies that forest vegetation employ to take up different forms of soil N, and the implications for forest management, remain insufficiently understood. To address this issue, we employed dual-labelled (13C-15N) tracers for three forms of available N (NH4+, NO3−, and glycine) in field experiments conducted in both natural and afforested stands in northeast China. As the growing season progressed, significant but non-uniform changes were observed in the N content, natural abundance of 15N in plant roots, and soil N properties, including soil NH4+, NO3−, amino acids, microbial biomass N, and the natural abundance of 15N. Whether in natural or artificial forests, the uptake rates and patterns of NH4+, NO3− and glycine by plant roots also varied, resulting in N niche differentiation among the coexisting plant species in terms of N form and timing. Principal component analysis and percentage similarity in N uptake patterns further revealed distinct N niche differentiation. Our results suggest that the uptake of different forms of soil N is likely driven by opportunistic rather than strictly preferential responses. This could provide an important basis for the N niche differentiation among coexisting plant species in the forest communities. Recognizing the temporal differentiation of N uptake niches during afforestation is essential to foster inter-specific coexistence, particularly in N-limited habitats. These findings provide critical insights for optimizing species composition in afforestation practices, thereby alleviating inter-specific competition, facilitating species coexistence, and maintaining productive forest ecosystems.