Prenatal alcohol exposure (EtOH) results in insulin resistance in rats of both genders with increased expression of hepatic gluconeogenic genes and glucose production. To investigate whether hepatic insulin signaling is defective, we studied 3 month-old female offspring of dams that were given EtOH during pregnancy, compared with those from pair-fed and control dams. We performed an intraperitoneal pyruvate tolerance test; we determined the phosphorylation status of hepatic phosphoinositide-dependent protein kinase-1 (PDK1), Akt and PKC before and after intravenous insulin bolus; and we measured mRNA and in vivo acetylation of TRB3 (tribbles 3) and PTEN (phosphatase and tensin homolog deleted on chromosome ten) as well as the expression of the histone acetylase (HAT) PCAF (p300/CREB-binding protein-associated factor), histone deacetylase (HDAC)-1, and HAT and HDAC activities. In EtOH compared with pair-fed and control offspring, basal and pyruvate-induced blood glucose was increased, insulin-induced PDK1, Akt, and PKC phosphorylation was reduced, PTEN and TRB3 expression was increased while their acetylation status was decreased in association with increased HDAC and decreased HAT activities. Thus, female adult rats prenatally exposed to EtOH have increased gluconeogenesis, reduced insulin signaling, and increased PTEN and TRB3 expression in the liver. In addition, PTEN and TRB3 are hypoacetylated, which can contribute to Akt inhibiting activity. These results suggest that hepatic insulin resistance in rats prenatally exposed to EtOH is explained, at least in part, by increased PTEN and TRB3 activity due to both increased gene expression and reduced acetylation.