Aging is a well-known risk factor for a large number of chronic diseases, including those of the lung. Cellular senescence is one of the hallmarks of aging, and contributes to the pathogenesis of age-related diseases. Recent studies implicate the reactive oxygen species (ROS)-generating enzyme, NADPH oxidase 4 (Nox4) in cellular senescence. In this study, we investigated potential mechanisms for epigenetic regulation of Nox4. We observed constitutively high levels of Nox4 gene/protein and activity in a model of replication-induced cellular senescence of lung fibroblasts. In replicative senescent fibroblasts, the Nox4 gene is enriched with the activation histone mark, H4K16Ac, and inversely associated with the repressive histone mark, H4K20Me3, supporting an active transcriptional chromatin conformation. Silencing of the histone acetyltransferase Mof, which specifically acetylates H4K16, down-regulates Nox4 gene/protein expression. The Nox4 gene promoter is rich in CpG sites; mixed copies of methylated and unmethylated Nox4 DNA were detected in both nonsenescent and senescent cells. Interestingly, the Nox4 gene is variably associated with specific DNA methyltransferases and methyl binding proteins in these two cell populations. These results indicate a critical role for histone modifications involving H4K16Ac in epigenetic activation of the Nox4 gene, while the role of DNA methylation may be contextual. Defining mechanisms for the epigenetic regulation of Nox4 will aid in the development of novel therapeutic strategies for age-related diseases in which this gene is overexpressed, in particular idiopathic pulmonary fibrosis and cancer.