To investigate N metabolism of two contrasting Populus species in acclimation to low N availability, saplings of slow-growing species (Populus popularis, Pp) and a fast-growing species (Populus alba × Populus glandulosa, Pg) were exposed to 10, 100, or 1000 μM NH4NO3. Despite greater root biomass and fine root surface area in Pp, lower net influxes of NH4 + and NO3 – at the root surface were detected in Pp compared to those in Pg, corresponding well to lower NH4 + and NO3 – content and total N concentration in Pp roots. Meanwhile, higher stable N isotope composition (δ15N) in roots and stronger responsiveness of transcriptional regulation of 18 genes involved in N metabolism were found in roots and leaves of Pp compared to those of Pg. These results indicate that the N metabolism of Pp is more sensitive to decreasing N availability than that of Pg. In both species, low N treatments decreased net influxes of NH4 + and NO3 –, root NH4 + and foliar NO3 – content, root NR activities, total N concentration in roots and leaves, and transcript levels of most ammonium (AMTs) and nitrate (NRTs) transporter genes in leaves and genes involved in N assimilation in roots and leaves. Low N availability increased fine root surface area, foliar starch concentration, δ15N in roots and leaves, and transcript abundance of several AMTs (e.g. AMT1;2) and NRTs (e.g. NRT1;2 and NRT2;4B) in roots of both species. These data indicate that poplar species slow down processes of N acquisition and assimilation in acclimation to limiting N supply.