The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and various stimuli associated with oxidative stress. Among the FOXO transcription factors, FOXO4 appears to be crucial in the response against oxidative stress. Therefore, it was the aim of this study to investigate the role of peroxide-induced oxidative stress and FOXO4 on PAI-1 expression under normoxia and hypoxia. Treatment of cells with hydrogen peroxide increased PAI-1 mRNA, protein, and promoter activity, and knocking down FOXO4 abolished the peroxide-dependent PAI-1 induction. PAI-1 promoter reporter gene assays revealed that the peroxide and FOXO4-dependent induction was mediated through the HIF-1 and CREB-binding HRE within the PAI-1 promoter. Western blot analyses then indicated that peroxide and FOXO4 downregulated HIF-1α levels, whereas CREB levels were increased. Chromatin immunoprecipitations showed that FOXO4 did not bind the PAI-1 promoter, whereas CREB binding was enhanced on FOXO4 overexpression. In addition, knockdown of CREB abolished the FOXO4-mediated PAI-1 induction. Together, these findings provide the first evidence that oxidative stress and FOXO4 induce PAI-1 expression through an indirect mechanism involving modulation of HIF-1α and CREB protein levels and that enhanced CREB binding to the PAI-1 promoter is critical for the PAI-1 induction under oxidative stress. Antioxid. Redox Signal. 13, 413–424.