Lipid metabolism influences membrane proteins, including ion channels, in health and disease. Fatty acid esters of CoA are important intermediates in fatty acid metabolism and lipid biosynthesis. In the present study, we examined the effect of acyl-CoAs on hSlo BK currents. Arachidonoyl-CoA (C(20)-CoA) induced beta2-dependent inhibition of hSlo-alpha current when applied intracellularly but not extracellularly. This action was also mimicked by other long-chain acyl-CoAs such as oleoyl-CoA (C(18)-CoA) and palmitoyl-CoA (C(16)-CoA), but not acetyl-CoA (C(2)-CoA, shorter chain), suggesting that the length of acyl chains, rather than CoA headgroups, is critical. When hSlo-alpha inactivation was induced by a free synthetic cationic beta2 NH2-terminus inactivation ball peptide, long-chain acyl-CoAs inhibited hSlo-alpha current and facilitated inactivation. The precursor fatty acids also facilitated the ball peptide-induced inactivation in a chain length-dependent manner, whereas sphingosine (positively charged) slowed this inactivation. When the beta2-induced inactivation was compared with that of the ball peptide, there was a negative shift in the steady state inactivation, slower recovery, and a reduced voltage-dependence of inactivation onset. These data suggest that electrostatic interactions with the cytosolic inactivation domain of beta2 mediate acyl-CoA modulation of BK currents. BK channel inactivation may be a specific target for lipid modulation in physiological and pathophysiological conditions.