Closure of pancreatic -cell ATP-sensitive potassium (K_ATP) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long chain acyl-CoA esters (acyl-CoAs) can potently activate K_ATP channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced -cell excitability and insulin secretion observed in obesity and type 2 diabetes (T2D). However, reduced -cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic -cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse -cells using virally-delivered over-expression of long chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on -cell K_ATP channel activity and cell excitability assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K_ATP channel activity in AdACSL-1-infected -cells cultured in media supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected -cells (+palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mM glucose. These results suggest a direct link between intracellular fatty ester accumulation and K_ATP channel activation, which may contribute to -cell dysfunction in type 2 diabetes.