Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. The HO products carbon monoxide and bilirubin not only provide antioxidant cytoprotection, but also have potent anti-inflammatory and immunomodulatory functions. Although HO-1 has previously been shown to be induced by various stimuli via activation of the p38 MAPK signaling pathway, the role of this protein kinase for HO-1 gene regulation is largely unknown. In the present study, it is demonstrated that pharmacological inhibitors of p38 induced HO-1 expression in monocytic cells. Moreover, basal HO-1 gene expression levels were markedly higher in untreated murine embryonic fibroblasts (MEF) from p38alpha(-/-) mice compared with those from wild-type mice. Transfection studies with luciferase reporter gene constructs indicate that increased HO-1 gene expression via inhibition of p38 was mediated by the transcription factor Nrf2, which is a central regulator of the cellular oxidative stress response. Accordingly, inhibitors of p38 induced binding of nuclear proteins to a Nrf2 target sequence of the HO-1 promoter, but did not affect HO-1 protein expression and promoter activity in Nrf2(-/-) MEF. Genetic deficiency of p38 led to enhanced phosphorylation of ERK and increased cellular accumulation of reactive oxygen species. In addition, pharmacological blockage of ERK and scavenging of reactive oxygen species with N-acetylcysteine reduced HO-1 gene expression in p38(-/-) MEF, respectively. Taken together, it is demonstrated that pharmacological inhibition and genetic deficiency of p38 induce HO-1 gene expression via a Nrf2-dependent mechanism in monocytic cells and MEF.