Plant mitochondria are important energy-producing structure and ROS are generated as byproducts. APX is one enzyme of the AsA-GSH cycle to reduces H2O2 to water. We identified both PtomtAPX and PtosAPX are located in mitochondria of Populus tomentosa Carr. PtomtAPX is specifically targeted to mitochondria, while PtosAPX is dual targeted to both chloroplast and mitochondria. The expression of PtomtAPX in mitochondria was 60-fold that of PtosAPX by ELISA and qPCR analysis. Under high light stress, the expression levels of PtosAPX increased, while that of PtomtAPX only slightly changed. Compared to the WT, the antisense transgenic PtomtAPX cell lines showed slowed growth, smaller cells impaired mitochondria in MS medium under normal growth. RNA-seq results showed 3121 genes significantly altered expression in the antisense cells, and most of them are important for mitochondrial function, particularly in oxidative phosphorylation. Our findings demonstrates a mitochondrial location for one APX isoform, and provide valuable insight into the mechanism which ROS balance is modulated by AsA-GSH cycle in mitochondria.