As glucose-induced insulin expression is mainly regulated at the translational level, and such regulation often involves the 5'-untranslated region (5'UTR), we examined the human proinsulin gene 5'UTR. RT-PCR and sequencing demonstrated that a proinsulin splice variant (SPV) generated from a cryptic 5'-splice site and retaining the first 26 bp of intron 1 was present in human pancreatic islets from normal donors. The expression of this SPV was metabolically regulated, as shown by quantitative real-time RT-PCR, revealing a more than 10-fold increase in the SPV in isolated human islets incubated at 16.7 mM compared with 1.67 mM glucose. In vitro wheat-germ translation and in vivom transfection studies demonstrated that the altered 5'UTR of the SPV increased translation. The SPV yielded 4-fold more in vitro translated preproinsulin protein than the native proinsulin mRNA, and the SPV 5'UTR inserted upstream from a luciferase reporter gene resulted in a more than 6-fold higher luciferase activity, suggesting enhanced translation in vivo. Retention of the 26 bp changed the proposed secondary RNA structure of the SPV, which may facilitate ribosomal binding and explain the increase in translation efficiency. These results suggest a novel mechanism by which metabolic changes can modulate the expression of 5'UTR SPVs and thereby regulate translation efficiency.