Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC‐3, DU‐145 and LNCaP) cells. 11,12‐Epoxyeicosatrienoic acid (11,12‐EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N‐methylsulfonyl‐6‐(2‐propargyloxyphenyl)hexanamide [MS‐PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration‐dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12‐EET‐induced cell migration. Importantly, synthetic EET antagonists (14,15‐epoxyeicosa‐5(Z)‐enoic acid [14,15‐EEZE], 14,15‐epoxyeicosa‐5(Z)‐enoic acid 2‐[2‐(3‐hydroxy‐propoxy)‐ethoxy]‐ethyl ester [14,15‐EEZE‐PEG] and 14,15‐epoxyeicosa‐5(Z)‐enoic‐methylsulfonylimide [14,15‐EEZE‐mSI]) inhibited EET‐induced cell invasion and migration. 11,12‐EET induced cell stretching and myosin‐actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15‐EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor‐mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer. (Cancer Sci 2010; 101: 2629–2636)