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Epigenetic alterations, including aberrant DNA methylation, contribute to tumor development and progression. Silencing of tumor suppressor genes may be ascribed to promoter DNA hypermethylation, a reversible phenomenon intensely investigated as potential therapeutic target. Previously, we demonstrated that eicosapentaenoic acid (EPA) exhibits a DNA demethylating action that promotes the re-expression of the tumor suppressor gene CCAAT/enhancer-binding protein δ (C/EBPδ). The C/EBPβ/C/EBPδ heterodimer formed appears essential for the monocyte differentiation commitment. The present study aims to evaluate the effect of EPA on RAS/extracellular signal regulated kinases (ERK1/2)/C/EBPβ pathway, known to be induced during the monocyte differentiation program. We found that EPA conditioning of U937 leukemia cells activated RAS/ERK/C/EBPβ pathway, increasing the C/EBPβ and ERK1/2 active phosphorylated forms. Transcriptional induction of the upstream activator H-Ras gene resulted in increased expression of H-Ras protein in the active pool of non raft membrane fraction. H-Ras gene analysis identified an hypermethylated CpG island in intron 1 that can affect the DNA-protein interaction modifying RNA polymerase II (RNAPII) activity. EPA treatment demethylated almost completely this CpG island, which was associated with an enrichment of active RNAPII. The increased binding of the H-Ras transcriptional regulator p53 to its consensus sequence within the intronic CpG island further confirmed the effect of EPA as demethylating agent. Our results provide the first evidence that an endogenous polyunsaturated fatty acid (PUFA) promotes a DNA demethylation process responsible for the activation of RAS/ERK/C/EBPβ pathway during the monocyte differentiation commitment. The new role of EPA as demethylating agent paves the way for studying PUFA action when aberrant DNA methylation is involved.