Blood ketone body levels increase during starvation and untreated diabetes. Here we tested the hypothesis that ketone bodies directly inhibit insulin action in skeletal muscle. We investigated the effect of d,l-beta-hydroxybutyrate (BOH; the major ketone body in vivo) on insulin-mediated glucose uptake (2-deoxyglucose) in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscle. BOH inhibited insulin-mediated glucose uptake in soleus (but not in EDL) muscle in a time- and concentration-dependent manner. Following 19.5 h of exposure to 5 mM BOH, insulin-mediated (20 mU/ml) glucose uptake was inhibited by approximately 90% (substantial inhibition was also observed in 3-O-methylglucose transport). The inhibitory effect of BOH was reproduced with d- but not l-BOH. BOH did not significantly affect hypoxia- or AICAR-mediated (activates AMP-dependent protein kinase) glucose uptake. The BOH effect did not require the presence/utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. To determine whether the BOH effect was mediated by oxidative stress, an exogenous antioxidant (1 mM tempol) was used; however, tempol did not reverse the BOH effect on insulin action. BOH did not alter the levels of total tissue GLUT4 protein or insulin-mediated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 but blocked insulin-mediated phosphorylation of protein kinase B by approximately 50%. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus ketone bodies may be potent diabetogenic agents in vivo.