The objective of this paper was to investigate the effects of berberine and mangiferin and their combination on glucose uptake in L6 myotubes and the mechanism.The L6 myotubes were cultured with serum-free HG-DMEM culture medium containing 2-deoxyglucose, and then berberine, mangiferin or berberine plus mangiferin (BM) with different concentrations were added into the culture medium resp.The level of phosphorylated AMPK was detected by Western blotting, and the results were statically analyzed.The relative efficacies of 0.625 μmol/L-1 berberine, mangiferin and BM to insulin were (91.26 ± 13.28)%, (61.34 ± 6.08)%, and (51.45 ± 7.94)%, resp., there was significant difference between BM and berberine (P < 0.05), and there was significant difference between BM and mangiferin (P < 0.01).The relative efficacies of 1.25 μmol/L-1 berberine, mangiferin and BM to insulin were (101.06 ± 4.57)%, (70.77 ± 6.43)%, and (52.85 ± 8.09)%, resp., there was remarkably significant difference between BM and berberine, and between BM and mangiferin (both P < 0.01).The relative efficacies of 2.5 μmol/L-1 berberine, mangiferin and BM to insulin were (102.13 ± 4.77)%, (81.09 ± 9.58)%, and (49.30 ± 5.47)%, resp., there was no significant difference between BM and berberine (P > 0.05), and there was remarkably significant difference between BM and mangiferin (P < 0.01).The relative efficacies of 5.0 μmol/L-1 berberine, mangiferin and BM to insulin were (120.47 ± 12.48)%, (98.66 ± 4.19)%, and (49.32 ± 2.20)%, resp., there was significant difference between BM and berberine (P < 0.05), and there was significant difference between BM and mangiferin (P < 0.01).1.25, 2.5 And 5.0 μmol/L-1 BM could improve the level of phosphorylated AMPK with concentration-dependent manner; 2.5, 5.0, and 10.0 berberine could μmol/L-1 improve the level of phosphorylated AMPK with concentration-dependent manner; while 1.0 and 2.0 μmol/L-1 could not improve the level of phosphorylated AMPK.Both BM and berberine could improve the glucose uptake and AMPK phosphorylation of L6 myotubes, and BM had good significant effect.