Heat shock transcription factors (HSFs) are crucial regulators in the responses of plants to heat stress, but their mechanisms of action in trees are unclear. We identified an HSFA gene, PeHSFA3, in Populus × euramericana (Populus deltoides × Populus nigra), which was highly responsive to heat. Its protein product was in both the cytoplasm and the nucleus. Stably overexpressing PeHSFA3 in poplar enhanced heat tolerance, improved photosynthetic capacity, increased antioxidant enzyme activities, reduced reactive oxygen species (ROS) levels, and upregulated antioxidant-related genes such as SODF2, SODCP, PER46–1, PER46–2, and PER31 under heat stress. Conversely, suppressing the PeHSFA3 gene by Virus-induced gene silencing (VIGS) in Populus × euramericana decreased its heat tolerance. qRT-PCR analysis demonstrated that PeHSFA3 enhances the expression of several heat-protective genes, including small heat-shock protein genes (HSP22C, HSP21–5, HSP18.1, HSP16A, HSP16B, HSP26.5–1, and HSP26.5–2) and ATG8. Transcriptomic analysis suggested that the activation of glutathione metabolic pathways contributes to PeHSFA3-mediated heat tolerance. Our findings reveal that PeHSFA3 mediates resistance to heat stress in poplar and could be used to enhance the thermotolerance of poplar.