The present study examined the acute effects of metformin on fatty acid (FA) metabolism in oxidative soleus (SOL) and glycolytic epitrochlearis (EPT) rodent muscle. Soleus and EPT were incubated for either 30 or 180 min in the absence or presence of 2 mM metformin, and with or without insulin (10 mU/mL). Metformin did not alter basal FA metabolism but countered the effects of insulin on FA oxidation and incorporation into triacylglyerol (TAG). Specifically, metformin prevented the insulin-induced suppression of FA oxidation in SOL, but did not alter FA incorporation into lipid pools. In contrast, in EPT, metformin blunted the incorporation of FA into TAG when insulin was present, but did not alter FA oxidation. In SOL, metformin resulted in a 50% increase in AMPK 2 activity and prevented the insulin-induced increase in malonyl CoA content. In both fiber types, basal and insulin-stimulated glucose oxidation was not significantly altered by metformin. All effects were similar regardless of whether measured following 30 or 180 min. Since increased muscle lipid storage and impaired FA oxidation have been associated with insulin resistance in this tissue, the ability of metformin to reverse these abnormalities in muscle FA metabolism may be a part of the mechanism by which metformin improves glucose clearance and insulin sensitivity. The present data also suggest that increased glucose clearance is not due to its' enhanced subsequent oxidation. Further studies are warranted to determine whether chronic metformin treatment has similar effects on muscle FA metabolism.