Metabolic regulation of pancreatic β-cell ATP-sensitive K⁺ channel (K_ATP channel) function plays a key role in the process of glucose-stimulated insulin secretion (GSIS). Modulation of K_ATP channel activity by long-chain acyl CoAs represents an important endogenous regulatory mechanism. Elevated acyl CoA levels have been reported in obese and type 2 diabetic individuals and may contribute to reduced β-cell excitability and impaired GSIS. Recent studies suggest that the composition of dietary fat may influence the effects of high-fat feeding on impaired GSIS. Therefore, we examined the effects of side-chain length and the degree of saturation of various acyl CoAs on K_ATP channel activity. Macroscopic currents from either wild-type or polymorphic (Kir6.2[E23K/I337V]) recombinant β-cell K_ATP channels were measured in inside-out patches by exposing the inner surface of the membrane to acyl CoAs at physiological nanomolar concentrations. Acyl CoAs increased both wild-type and polymorphic K_ATP channel activity with the following rank order of efficacy: C18:0, C18:1trans ∼ C18:1cis, C20:4 = C16:0, C16:1, and C18:2. A significant correlation exists between activation and acyl CoA hydrophobicity, suggesting that both side-chain length and degree of saturation are critical determinants of K_ATP channel activation. Our observations reveal a plausible mechanism behind the disparate effects of acyl CoA saturation on K_ATP channel activation and suggest that dietary fat composition may determine the severity of impaired GSIS via differential activation of β-cell K_ATP channels.