Scope: Eicosapentaenoic acid (EPA), abundant in oily fish, is reported to reduce skin inflammation and provide photoprotection, potential mechanisms include competition with arachidonic acid (AA) for metabolism by cyclooxygenases/lipoxygenases to less pro-inflammatory mediators. We thus examine impact of EPA intake on levels of AA, EPA and their resulting eicosanoids in human skin with or without ultraviolet radiation (UVR) challenge. Methods and results: In a double-blind randomised controlled study, 79 females took 5 g EPA-rich or control lipid for 12 wk. Pre- and post-supplementation, red blood cell and skin polyunsaturated fatty acids were assessed by GC, and eicosanoids from unexposed and UVR-exposed skin by LC-MS/MS. Active supplementation increased red blood cell and dermal EPA versus control (both p <0.001), lowering relative AA:EPA content (4:1 versus 15:1 and 5:1 versus 11:1, respectively; both p <0.001). Pre-supplementation, UVR increased PGE2, 12-hydroxyeicosatetraenoic acids, 12-HEPE (all p <0.001) and PGE3 (p <0.05). Post-EPA, PGE2 was reduced in unchallenged skin (p <0.05) while EPA-derived PGE3 (non-sign) and 12-HEPE (p <0.01) were elevated post-UVR. Thus, post-EPA, PGE2:PGE3 was lower in unchallenged (12:1 versus 28:1; p <0.05) and UVR exposed (12:1 versus 54:1; p <0.01) skin; 12-hydroxyeicosatetraenoic acids:12-HEPE was lower in UVR-exposed skin (3:1 versus 11:1; p <0.001). Conclusion: Dietary EPA augments skin EPA:AA content, shifting eicosanoid synthesis towards less pro-inflammatory species, and promoting a regulatory milieu under basal conditions and in response to inflammatory insult. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.