The sodium-calcium exchanger isoform 1 (NCX1) is intimately involved in the regulation of calcium (Ca²⁺) homeostasis in many tissues including excitation-secretion coupling in pancreatic -cells. Our group has previously found that intracellular long chain acyl CoAs are potent regulators of the cardiac NCX1.1 splice variant. In spite of this, little is known about the biophysical properties of -cell NCX1 splice variants and the effects of intracellular modulators on their important physiological function in health and disease. Here, we show that the forward-mode activity of –cell NCX1 splice variants is differentially modulated by acyl CoAs and is dependent both upon the intrinsic biophysical properties of the particular NCX1 splice variant as well as the side chain length and degree of saturation of the acyl CoA moiety. Notably, saturated long chain acyl CoAs increased both peak and total NCX1 activity, whereas polyunsaturated long chain acyl CoAs did not show this effect. Furthermore, we have identified the exon within the alternative splicing region that bestows sensitivity to acyl CoAs. We conclude that the physiologically relevant forward-mode activity of NCX1 splice variants expressed in the pancreatic -cell are sensitive to acyl CoAs of different saturation and alterations in intracellular acyl CoA levels may ultimately lead to defects in Ca²⁺-mediated exocytosis and insulin secretion.