Glucose-induced insulin release from pancreatic beta-cells relies largely on glucose metabolism and mitochondrial ATP synthesis. Inhibiting the mitochondrial Na(+)/Ca(2+) exchanger (mNCE) using 7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157) has been suggested to enhance ATP synthesis and insulin secretion from rat islets by promoting mitochondrial Ca(2+) accumulation. In this study we examined the effects of CGP-37157 on human and mouse islet cells. Surprisingly, we found that insulin secretion from perifused islets was reduced by CGP-37157. Cytosolic Ca(2+) measurements revealed that CGP-37157 dose-dependently blocked glucose- and KCl-stimulated Ca(2+) signals in both human and mouse beta-cells. Conversely, CGP-37157 induced mitochondrial hyperpolarization, NAD(P)H rises, and triggered diazoxide- and nifedipine-sensitive cytosolic Ca(2+) transients in a subset of quiescent cells bathed in sub-stimulatory glucose, which is in accord with metabolic activation by the compound. Hence, while blocking mNCE with CGP-37157 may augment metabolism of human and mouse beta-cells, the propagation of metabolic signals is hampered by simultaneous inhibition of voltage-gated Ca(2+) influx, and ultimately insulin secretion. Efforts to use CGP-37157 or design related compounds for therapeutic purposes should take these competing effects into account.