Background. The aim of this study was to assess the effects of low concentrations of H2O2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H2O2. Flow cytometric analysis was used to select an optimal concentration of H2O2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H2O2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H2O2. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H2O2; in contrast, ERK5 knock-down abrogated the effects of H2O2. Conclusions. Our results confirmed that low concentrations of H2O2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.