Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression.