In this study, we examined whether adenoviral-mediated glycerol kinase (AdV-CMV-GlyK) expression in isolated rat pancreatic islets could introduce glycerol-induced proinsulin biosynthesis. In AdV-CMV-GlyK–infected islets, specific glycerol-induced proinsulin biosynthesis translation and insulin secretion were observed in parallel from the same islets. The threshold concentration of glycerol required to stimulate proinsulin biosynthesis was lower (0.25–0.5 mmol/l) than that for insulin secretion (1.0–1.5 mmol/l), reminiscent of threshold differences for glucose-stimulated proinsulin biosynthesis versus insulin secretion. The dose-dependent glycerol-induced proinsulin biosynthesis correlated with the rate of glycerol oxidation in AdV-CMV-GlyK–infected islets, indicating that glycerol metabolism was required for the response. However, glycerol did not significantly increase lactate output from AdV-CMV-GlyK–infected islets, but the dihydroxyacetone phosphate (DHAP) to α-glycerophosphate (α-GP) ratio significantly increased in AdV-CMV-GlyK–infected islets incubated at 2 mmol/l glycerol compared with that at a basal level of 2.8 mmol/l glucose (P ≤ 0.05). The DHAP:α-GP ratio was unaffected in AdV-CMV-GlyK–infected islets incubated at 2 mmol/l glycerol in the added presence of α-cyanohydroxycinnaminic acid (α-CHC), an inhibitor of the plasma membrane and mitochondrial lactate/pyruvate transporter. However, α-CHC inhibited glycerol-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK–infected islets (>75%; P = 0.05), similarly to glucose-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK–infected and control islets. These data indicated that in AdV-CMV-GlyK–infected islets, the importance of mitochondrial metabolism of glycerol was required to generate stimulus–response coupling signals to induce proinsulin biosynthesis and insulin secretion.