Endothelial progenitor cells (EPCs) repair vascular damage and participate in neovascularization. Accumulating evidence has demonstrated that EPCs have therapeutic potential in reactive oxygen species (ROS)-mediated vascular diseases. In this study, to investigate the effects of oxidative stress on EPCs, EPCs were treated with H2O2 at different final concentrations for 3 h. MTT assay, scratch-wound assay and Matrigel invasion assay revealed that cell proliferation, migration and tubule formation and function, respectively, were impaired under H2O2 stress in a concentration-dependent manner. To determine protein response to H2O2 stress, two-dimensional differential in-gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry were performed. The results revealed that triosephosphate isomerase and ADP-sugar pyrophosphatase were downregulated, while peroxiredoxin-2, thioredoxin-dependent peroxide reductase, mitochondrial (Prx‑3), peroxiredoxin-6, EGF-containing fibulin-like extracellular matrix protein 1, vimentin and Rab GDP dissociation inhibitor α were upregulated in the H2O2-treated EPCs. To further confirm the results from mass spectrometry, the expression pattern of Prx-3 in response to H2O2 stress was examined by western blot analysis. The data presented in this study provide novel insight into the defensive mechanisms of EPCs and the pathways of oxidative damage in an oxidative environment.