Objective To investigate whether berberine exerts hypoglycemic effect in dependence of adenosine 5￠-monophosphate-activated protein kinase (AMPK) signal pathway. Methods After human liver hepatocellular cell line HepG2 and mouse myoblast cell line C2C12 were treated with berberine at different concentrations, glucose consumption and lactate production were used to evaluate the glucose-lowering and anaerobic glycolysis effects of berberine. Compound C (CC), an AMPK inhibitor, and recombinant adenovirus Ad-DN-AMPK expressing dominant negative mutant of AMPK, were used to inhibit the expression and block the activity of AMPK in the cells. AMPK phosphorylation and acetyl coA carboxylase (ACC) phosphorylation were measured with Western blotting to assess the activity of AMPK pathway. Results Berberine significantly increased glucose consumption and lactate production in HepG2 and C2C12 cells in a dose-dependent manner. AMPK and ACC phosphorylation were significantly stimulated by 5 and 10μmol/L berberine. CC and adenovirus-dominant negative-AMPK (Ad-DN-AMPK) adenovirus obviously inhibited AMPK activity, but failed to diminish the promotion of glucose utilization and lactate production induced by berberine. Conclusion Berberine significantly enhances glucose consumption by stimulating glucose metabolism, in absence of AMPK pathway activation. Therefore, even in the case of the expression or activity of AMPK is inhibited, berberine is still able to exert a significant hypoglycemic effect.