We have reported an early decrease of glycemia in rats fed a biotin-deficient diet, with reduced cellular ATP levels and suggesting increased insulin sensitivity. Here we show that biotin deprived rats are more tolerant to glucose as shown by both oral and intraperitoneal glucose tolerance tests, during which insulin plasma levels were significantly diminished in deficient rats compared to controls. Biotin-deficient rats had lower blood glucose concentrations during intraperitoneal insulin sensitivity tests than controls. Furthermore, more glucose was infused to maintain euglycemia in the biotin deficient rats during hyperinsulinemic euglycemic clamps studies. These results demonstrate augmented sensitivity to insulin in biotin-deprived rats. They are most likely consequence of an insulin-independent effect of AMPK activation on GLUT4 membrane translocation with increased glucose uptake. In biotin deficient cultured L6 muscle cells, there was increased phosphorylation of the energy sensor AMPK. We have now confirmed the augmented AMPK activation in both biotin-deprived in vivo muscle and in cultured muscle cells. In these cells, glucose uptake is increased by AMPK activation by AICAR, and is diminished by its knockdown by the specific siRNAs directed against its a1 and a2 catalytic subunits; all of these effects being largely independent of the activity of the insulin signaling pathway that was inhibited with Wortmannin. The enhanced insulin sensitivity in biotin deficiency likely has adaptive value for organisms due to the hormone promotion of uptake and utilization not only of glucose, but other nutrients such as branched-chain amino acids, whose deficiency has been reported to increase insulin tolerance.