Background: Metformin-induced activation of AMPK has been associated with enhanced glucose uptake in skeletal muscle but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent upon AMPK signaling. Methods: Oral doses of metformin or saline treatment were given muscle-specific kinase α2 dead AMPK mice (KD) and wild type (WT) littermates either once or chronically for 2 weeks. Soleus and Extensor Digitorum Longus (EDL) muscles were used for measurements of glucose transport and Western blot analyzes. Results: Chronic treatment with metformin enhanced insulin-stimulated glucose uptake in soleus muscles of WT (45%, P<0.01), but not in AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr308 and Ser473 phosphorylation was not changed by metformin treatment. Downstream of Akt, TBC1D4 Thr642 and Ser711 phosphorylation and Rab4 and GLUT4 protein expressions were not affected by metformin treatment. Also AMPK catalytic subunit, TBC1D4 and hexokinase II proteins were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. Conclusion: We provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in skeletal muscle in response to chronic metformin treatment.