5’-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion such as hypoxia or inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for the stimulation of glucose transport that occurs in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Clone 9 cells (expressing Glut1) with sodium azide (5 mM for 2 h) or with the AMPK activator, 5’-aminoimidazole-4carboxamide-1--D-ribofuranoside (AICAR) (2 mM for 2 hours), stimulated the rate of glucose transport by 2- to 4-fold. Employing small interference RNA (siRNA) directed against AMPK1 isoform alone or against both 1 and 2 isoforms resulted in a significant inhibition of the glucose transport response and the content of P-AMPK1&2 and P-ACC in response to azide. Transfection with siRNA directed against AMPK2 alone did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response, and abolished the increase in P-AMPK1&2 in cells treated with azide or hypoxia. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of co-immunoprecipitation of P-AMPK1&2 with Glut1. However, LKB1 was found to be associated with P-AMPK1&2. We conclude that activation of AMPK plays a sufficient and necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.