We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca2+-activated K+ (BKCa) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BKCa channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, Nω-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BKCa activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BKCa channel activity in IR myocytes, whereas in the I/O configuration, BKCa channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through KCa channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BKCa channel.