Abstract: Objective To determine the expression level of micro non-coding RNA-133b (miR-133b) in the liver of different mouse model and its effect of miR-133b on gluconeogenesis in Huh7 hepatocytes. Methods The expression of miR-133b in the livers of wild type C57BL/6 mice fed with high fat diet, wild type C57BL/6 mice fasted for 24 h and diabetes animal model db/db mice were detected by real-time polymerase chain reaction (RT-PCR). After miR-133b mimic or inhibitor was transfected into Huh7 hepatocytes, glucose production experiment was conducted and mRNA and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were measured. Results The expression of miR-133b in the liver of wild-type C57BL/6 mice fed with high-fat diet was significantly increased, while that in the liver of wild-type C57BL/6 mice and db/db mice fasted for 24 h was significantly decreased. Overexpression of miR-133b in Huh7 hepatocytes significantly reduced the level of gluconeogenesis in Huh7 hepatocytes, while inhibition of miR-133b expression significantly increased the level of gluconeogenesis in Huh7 hepatocytes. Conclusions miR-133b is closely related to liver nutrient metabolism and it can regulate the glycogenesis of hepatocytes by regulating the expression level of Pepck gene.