Biotin is known to influence hepatic glucokinase (GK) expression both at a transcriptional and at a translational level. The aim of the present paper was to investigate the effect of biotin on pancreatic GK. For this purpose, RIN1046-38 cells were cultured in the presence of different biotin concentrations for different times; there-after, GK mRNA expression, GK activity and insulin release were studied. Results demonstrated that biotin has a biphasic effect on GK mRNA expression, being stimulatory after short-term treatment and inhibitory after longterm treatment. GK activity was increased after long-term treatment. Insulin release was not affected by biotin treatment. These data suggest that biotin may influence glucose metabolism also by acting directly at the level of beta-cells.