Insulin production in pancreatic β cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40–48 bp from the 5′ end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5′UTR, the human ppI 5′UTR conferred glucose regulation of translation. However alternative splicing of the human 5′UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.