AKT is a serine–threonine protein kinase that plays important roles in cell growth, proliferation and apoptosis. It is activated after binding to phosphatidylinositol phosphates (PIPs) with phosphate groups at positions 3,4 and 3,4,5 on the inositol ring. In spite of extensive research on AKT, one aspect has been largely overlooked, namely the role of the fatty acid chains on PIPs. PIPs are phospholipids composed of a glycerol backbone with fatty acids at the sn -1 and sn -2 position and inositol at the sn -3 position. Here, we show that polyunsaturated fatty acids (PUFAs) modify phospholipid content. Docosahexaenoic acid (DHA), an ω3 PUFA, can replace the fatty acid at the sn -2 position of the glycerol backbone, thereby changing the species of phospholipids. DHA also inhibits AKT ^T308 but not AKT ^S473 phosphorylation, alters PI(3,4,5)P ₃ (PIP ₃ ) and phospho-AKT ^S473 protein localization, decreases pPDPK1 ^S241 -AKT and AKT–BAD interaction and suppresses prostate tumor growth. Our study highlights a potential novel mechanism of cancer inhibition by ω3 PUFA through alteration of PIP ₃ and AKT localization and affecting the AKT signaling pathway.