The stomata regulate CO2uptake and efficient water usage, thereby promoting drought stress tolerance. NAC proteins (NAM, ATAF1/2, and CUC2) participate in plant reactions following drought stress, but the molecular mechanisms underlying NAC-mediated regulation of stomatal movement are unclear. In this study, a novel NAC gene from Reaumuria trigyna, RtNAC055, was found to enhance drought tolerance via a stomatal closure pathway. It was regulated by RtMYC2 and integrated with jasmonic acid signaling and was predominantly expressed in stomata and root. The suppression of RtNAC055could improve jasmonic acid and H2O2production and increase the drought tolerance of transgenic R. trigynacallus. Ectopic expression of RtNAC055in the Arabidopsis atnac055mutant rescued its drought-sensitive phenotype by decreasing stomatal aperture. Under drought stress, overexpression of RtNAC055in poplar promoted ROS (H2O2) accumulation in stomata, which accelerated stomatal closure and maintained a high photosynthetic rate. Drought upregulated the expression of PtRbohD/F, PtP5CS2, and PtDREB1.1, as well as antioxidant enzyme activities in heterologous expression poplars. RtNAC055 promoted H2O2production in guard cells by directly binding to the promoter of RtRbohE, thus regulating stomatal closure. The stress-related genes RtDREB1.1/P5CS1were directly regulated by RtNAC055. These results indicate that RtNAC055 regulates stomatal closure by maintaining the balance between the antioxidant system and H2O2level, reducing the transpiration rate and water loss, and improving photosynthetic efficiency and drought resistance.