A loss-of-function mutation of the mitochondrial beta-oxidation enzyme L-3-hydroxyacyl-coA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinaemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial beta-oxidation. On the other hand, beta cells expressed relatively low levels of other beta-oxidation enzymes (ACADS, ACADM, ACADL, ACAA2). HADHSC expression was sequence-specifically silenced by RNA interference , and the effects examined on glucose-stimulated insulin secretion (GSIS) following 48 to 72h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of L-3-hydroxyacyl-coA dehydrogenase, short-chain (HADHSC) and a low expression of other beta-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.