Thiamin (vitamin B₁) is known to activate carbohydrate metabolism in part through activation of pyruvate dehydrogenase. The purpose of this study was to investigate the effect of thiamin tetrahydrofurfuryl disulfide (TTFD), a thiamin derivative, on utilization of exogenous glucose by measuring oxidation of ¹³C-glucose at rest and during prolonged exercise in mice under normal dietary conditions. Mice orally ingested TTFD (0.1 mg/g BW [body weight]) and ¹³C-glucose (0.8 mg/g BW) or ¹³C-lactate (0.1 mg/g BW) plus glucose (0.8 mg/g BW) at rest or before endurance running. The average percent of ¹³C atoms in total ¹²C+¹³C (¹³C atom%) in expired air after ingestion of ¹³C-glucose at rest was significantly lower in the TTFD group than in the control group. No significant difference was found in ¹³C atom% in expired air after ingestion of ¹³C-glucose and prolonged exercise. In addition, no significant effect of TTFD was found in expired ¹³C atom% after ingestion of ¹³C-lactate plus glucose at rest. TTFD also had no effect on concentrations of muscle or liver glycogen at rest. These results suggest that TTFD, which is a thiamin derivative, decreases oxidation of exogenous glucose at rest, but not during exercise.