Although the cyclic AMP response element–binding protein (CREB) plays an important role in the survival of neuronal cells and T lymphocytes, the role of CREB in vascular smooth muscle cells (VSMCs) is incompletely characterized. We examined the role of CREB in VSMCs stimulated with reactive oxygen species. Activation of CREB was examined by Western blot analysis with an antibody that specifically recognizes phosphorylation at serine 133 of CREB, which is a critical marker of activation. Hydrogen peroxide (H₂O₂) time-dependently induced phosphorylation of CREB, with a peak at 15 minutes. The H₂O₂-induced phosphorylation of CREB was partially blocked by inhibition of either extracellular signal–regulated protein kinase kinase by PD98059 or of p38 mitogen-activated protein kinase (MAPK) by SB203580. AG1478, an epidermal growth factor receptor (EGFR) inhibitor, suppressed the H₂O₂-induced phosphorylation of CREB and tyrosine phosphorylation of EGFR. Overexpression of the dominant-negative form of CREB by an adenovirus vector suppressed H₂O₂-induced c-fos expression. These findings suggest that H₂O₂ induces CREB phosphorylation through EGFR transactivation and mitogen-activated protein kinase pathways. CREB might be a novel redox-sensitive transcription factor involved in the regulation of VSMC gene expression.