In the present study, the effects of nitrogen (N) supply on water uptake, drought resistance, and hormone regulation were investigated in Populus simonii seedlings grown in hydroponic solution with 5% polyethylene glycol (PEG)-induced drought stress. While acclimating to drought, the P. simonii seedlings exhibited a reduction in growth; differential expression levels of aquaporins (AQPs); activation of auxin (IAA) and abscisic acid (ABA) signaling pathways; a decrease in the net photosynthetic rate and transpiration rate; and an increase in stable nitrogen isotope composition (δ¹⁵N), total soluble substances, and intrinsic water use efficiency (WUEi), with a shift in the homeostasis of reactive oxygen species (ROS) production and scavenging. A low N supply (0.01 mM NH₄NO₃) or sufficient N supply (1 mM NH₄NO₃) exhibited distinct morphological, physiological, and transcriptional responses during acclimation to drought, primarily due to strong responses in the transcriptional regulation of genes encoding AQPs; higher soluble phenolics, total N concentrations, and ROS scavenging; and lower transpiration rates, IAA content, ABA content, and ROS accumulation with a sufficient N supply. P. simonii can differentially manage water uptake and hormone modulation in response to drought stress under deficient and sufficient N conditions. These results suggested that increased N may contribute to drought tolerance by decreasing the transpiration rate and O₂⁻ production while increasing water uptake and antioxidant enzyme activity.