Chloroplasts are the most major producers of reactive oxygen species (ROS) during photosynthesis. However, the function of thylakoid ascorbate peroxidase (tAPX) in response to oxidative stress in wood trees is largely unknown. Our results showed that PtotAPX of Populus tomentosa could effectively utilize ascorbic acid (AsA) to hydrolyze hydrogen peroxide (H₂O₂) in vitro. The overexpression or antisense of PtotAPX (OX-PtotAPX or anti-PtotAPX, respectively) in Populus tomentosa plants did not significantly affect plant morphology during plant growth. When treated with methyl viologen (MV), the OX-PtotAPX plants exhibited less morphological damage under stress conditions compared to WT plants. OX-PtotAPX plants maintained lower H₂O₂ levels and malondialdehyde (MDA) contents, but more reduced AsA levels, a higher photosynthetic rate (Pn), and the maximal photochemical efficiency of PSII (Fv/Fm), whereas anti-PtotAPX plants showed the opposite phenotype. Furthermore, the activity of APX was slightly higher in OX-PtotAPX under normal growth conditions, and this activity significantly decreased after stress treatment, which was the lowest in anti-P. Based on these results, we propose that PtotAPX is important for protecting the photosynthetic machinery under severe oxidative stress conditions in P. tomentosa, and is a potential genetic resource for regulating the stress tolerance of woody plants.