Glucose transport in skeletal muscle can be mediated by two separate pathways, one stimulated by insulin and the other by muscle contraction. High-fat feeding impairs glucose transport in muscle, but the mechanism remains unclear. FVB mice (3 weeks old) were fed a high-fat diet (55% fat, 24% carbohydrate, 21% protein) or standard chow for 3–4 weeks or 8 weeks. Insulin-stimulated glucose transport, assessed with either 2-deoxyglucose or 3-O-methylglucose was decreased 35–45% (P < 0.001) in isolated soleus muscle, regardless of diet duration. Similarly, glucose transport stimulated by okadaic acid, a serine/threonine phosphatase inhibitor, was also 45% lower with high-fat feeding, but the glucose transport response to hypoxia or N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) (which are stimulators of the “contraction pathway”) was intact. Hexokinase I, II, and total activity were normal in soleus muscle from highfat–fed mice. GLUT4 expression in soleus muscle from the high-fat–fed mice was also normal, but the insulinstimulated cell surface recruitment of GLUT4 assessed by exofacial photolabeling with [³H]-ATB bismannose was reduced by 50% (P < 0.001). Insulinreceptor substrate 1 (IRS-1) associated phosphatidylinositol (PI) 3-kinase activity stimulated by insulin was also reduced by 36% (P < 0.001), and expression of p85 and p110_b subunits of PI 3-kinase was normal. In conclusion, high-fat feeding selectively impairs insulin-stimulated, but not contraction-pathway–mediated, glucose transport by reducing GLUT4 translocation to the plasma membrane. This appears to result from an acquired defect in insulin activation of PI 3-kinase. Since effects of okadaic acid on glucose transport are independent of PI 3-kinase, a second signaling defect may also be induced.