Insulin promotes glucose consumption via regulation of miR-99a/mTOR/PKM2 pathway. Insulin is known to regulate multiple cellular functions and is used for the treatment of diabetes. MicroRNAs have been demonstrated to be involved in many human diseases, including Type 2 diabetes. In this study, we showed that insulin decreased miR-99a expression levels, but induced glucose consumption and lactate production, and increased the expression of mTOR, HIF-1alpha and PKM2 in HepG2 and HL7702 cells. Forced expression of miR-99a or rapamycin treatment blocked insulin-induced PKM2 and HIF-1alpha expression, and glucose consumption and lactate production. Meanwhile, knockdown of HIF-1alpha inhibited PKM2 expression and insulin-induced glucose consumption. Taken together, these findings will reveal the role and mechanism of insulin in regulating glycolytic activities via miR-99a/mTOR .