Biotin functions as a coenzyme for four carboxylases involved in energy metabolism in mammals. Besides these classical functions, biotin has novel functions in the cellular processes via the modulation of gene expression. In this study, we examined the alteration of gene expression by biotin administration in the liver of streptozotocin (STZ)-induced diabetic rats. In comparison with the control, the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were significantly reduced and glucokinase mRNA was increased 3 h after the administration of biotin or insulin. The expression of hepatocyte nuclear factor 4alpha, one of the transcription factors responsible for gluconeogenic gene expression, was decreased by biotin at both mRNA and protein levels. In addition, forkhead box O1 and sterol regulatory element-binding protein 1c mRNA expression that was enhanced by the insulin treatment was inversely decreased by biotin. These results indicate that biotin repressed the gluconeogenic genes and their transcription factors via a pathway independent of insulin-signaling and could improve the diabetic condition.