Although it is understood that hydrogen peroxide (H2O2) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H2O2 in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H2O2. The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0 ± 4.7 h versus 28.6 ± 0.8 h, p<0.05), consistent with a diminished growth rate and H2O2 release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G0/G1 phase in hCatTg MAECs compared to the wild-type cells (25.0 ± 0.9 h versus 15.9 ± 1.4 h, p < 0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G0/G1- to S-phase transition in the cell cycle, including the cyclin D–Cdk4 and cyclin E–Cdk2 complexes. Moreover, the reduction in cyclin–Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G0/G1- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H2O2 scavenger, suggest that endogenously produced H2O2 mediates MAEC proliferation by fostering the transition from G0/G1 to S phase.