A member of APX (ascorbate peroxidase) gene superfamily of Populus tomentosa was cloned from the cDNA of reverse transcribed RNA. The open reading frame (ORF) of PtAPX2 was 864 bp in length, and encoded a protein containing 287 amino acids with a molecular weight of 3178 ku. The carboxyl terminal of PtAPX2 contained a putative transmembrane domain, which might be responsible for its peroxisomal subcellular localization. In order to gain insights into the characteristics of this enzyme, the PtAPX2 protein was expressed in Escherichia coli and purified to homogeneity. Its enzyme activity was assayed in vitro. The Km values to substrate ascorbate and H2O2 were determined to be (1.37±0.22) and (0.026±0.003) mmol/L, respectively. The Vmax values to substrate ascorbate and H2O2 were (3.95±0.46) and (1.27±0.03) mmol/(L•min•mg), respectively. PtAPX2 exhibited the most potent activity at 28 ℃, pH 7.0-7.4. Quantitative assays using fluorescent realtime PCR revealed the highest transcription level of PtAPX2 in the mesophyll of aged leaves. The results of the prediction of subcellular localization, the pattern of substrate binding, enzyme kinetics and tissuespecific expression of PtAPX2 together expand our knowledge on the mechanism underlying oxidative stresses resistance in woody plants.