【Objective】 Apyrases are key enzymes that hydrolyze nucleoside di- and triphosphates inside or outside the cell, which play crucial roles in regulating plant growth and stress responses. This study identified the molecular function of Apyrase gene PagAPY1 in Populus alba × P. glandulosa and elucidated the regulatory mechanisms of PagAPY1 in enhancing drought tolerance in poplars. 【Method】 Using bioinformatics approaches, the PagAPY1 gene was identified in genome of P. alba × P. glandulosa. A PagAPY1-YFP fusion protein was constructed for subcellular localization analysis. PagAPY1-overexpressing transgenic poplars were obtained through agrobacterium-mediated transformation. qRT-PCR was used to detect the expression of PagAPY1 and other stress-related genes in both wild type and transgenic poplars. The growth phenotype, root development, and leaf water retention were analyzed under drought stress. Leave stomatal aperture was measured in both WT and transgenic poplars under drought or ABA treatment. 【Result】 The protein coded by PagAPY1 possessed conserved ACR domains and amino acid residues critical for enzyme catalytic activity. PagAPY1 was localized to the Golgi apparatus, where it could participate in the hydrolysis of NTP/NDP. The expression of PagAPY1 was induced by drought. Eight PagAPY1-overexpressing transgenic poplars were identified and three OE lines with the highest PagAPY1 expression levels were selected for further experiments. Under drought stress, the PagAPY1-OE transgenic lines showed improved survival rates, plant height, root length, root biomass compared with the wild-type poplars. Under osmotic stress, the transgenic plants exhibited greater adventitious root growth compared with the wild type. Meanwhile, the stomatal aperture was more significantly decreased in the transgenic lines during drought condition. In contrast to wild-type, the OE lines exhibited greater sensitivity to ABA-induced stomatal closure. The expression of genes involved in ROS signaling and genes coding antioxidant enzymes was significantly upregulated in the PagAPY1-overexpressing lines under drought condition. 【Conclusion】 Under drought stress, PagAPY1 facilitates auxin polar transport by maintaining protein glycosylation and eATP homeostasis, thereby enhances root growth in the overexpression poplars. Meanwhile, through regulating eATP levels in leaves, PagAPY1 promotes stomatal closure to reduce leaf water loss under drought stress. PagAPY1-overexpressing also increases the expression levels of genes that are involved in ROS signaling and antioxidant enzyme system, thereby enhancing drought resistance in poplars.