Adverse events during pregnancy, including prenatal ethanol (EtOH) exposure, are associated with insulin resistant diabetes in male rat offspring, but it is unclear whether this is true for female offspring. We investigated whether prenatal EtOH exposure alters glucose metabolism in adult female rat offspring and whether this is associated with reduced in vivo insulin signaling in skeletal muscle. Female Sprague Dawley rats were given EtOH, 4g/kg/day by gavage throughout pregnancy. Glucose tolerance test and hyperinsulinemic euglycemic clamp were performed, and insulin signaling was investigated in skeletal muscle, in adult female offspring. We gave insulin intravenously to these rats and determined the association of glucose transporter-4 with plasma membranes, and the phosphorylation of phosphoinositide-dependent protein kinase-1 (PDK1), Akt and PKC. Although EtOH offspring had normal birth weight, they were overweight as adults and had fasting hyperglycemia, hyperinsulinemia and reduced insulin-stimulated glucose uptake. Following insulin treatment, EtOH-exposed rats had decreased membrane glucose transporter-4, PDK1, Akt and PKC in the gastrocnemius muscle, compared with control rats. Insulin-stimulation of PDK1, Akt and PKC phosphorylation was also reduced. In addition, the expression of the protein tribbles-3 and the phosphatase enzyme activity of PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome Ten), which prevent Akt activation, were increased in muscle from EtOH-exposed rats. Female rat offspring exposed to EtOH in utero develop insulin resistant diabetes in association with excessive PTEN and tribbles-3 signaling downstream of the phosphatidylinositol 3-kinase pathway in skeletal muscle, which may be a mechanism for the abnormal glucose tolerance.