Helicobacter pylori is an important risk factor for gastric inflammation, which is mediated by multiple signaling pathways. The aim of this study was to investigate the effects of polyunsaturated fatty acids (PUFAs), such as linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA), on the expression of the proinflammatory chemokine interleukin-8 (IL-8) in H. pylori-infected gastric epithelial AGS cells. To investigate whether PUFAs modulate H. pylori-induced inflammatory signaling, we determined the activation of epidermal growth factor receptor (EGFR), protein kinase C-δ (PKC δ), mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF- κB), and activator protein-1 (AP-1) as well as IL-8 expression in H. pylori-infected gastric epithelial cells that had been treated with or without PUFAs. We found that PUFAs inhibited IL-8 mRNA and protein expression in H. pylori-infected cells. ω-3 fatty acids (ALA, and DHA) suppressed the activation of EGFR, PKC δ, MAPK, NF- κB, and AP-1 in these infected cells. LA did not prevent EGFR transactivation and exhibited a less potent inhibitory effect on IL-8 expression than did ALA and DHA. In conclusion, PUFAs may be beneficial for prevention of H. pylori-associated gastric inflammation by inhibiting proinflammatory IL-8 expression.