Epidemiological and experimental studies have demonstrated that early postnatal nutrition has been associated with the long-term effects on glucose homeostasis in adulthood. Recently, our group demonstrated that undernutrition during early lactation affects the expression and activation of key proteins of the insulin signaling cascade in rat skeletal muscle during post-natal development. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity in peripheral tissues, we investigated the insulin signaling in adipose tissue. Adipocytes were isolated from epididimal fat pads of adult male rats which were the offspring of dams fed either a normal or a protein-free diet during the first 10 days of lactation. The cells were incubated with 100 nM of insulin before the assays for immunoblotting analysis, 2-deoxiglucose uptake, immunocytochemistry for GLUT-4, and/or actin filaments. Following insulin stimulation, adipocytes isolated from undernourished rats presented reduced tyrosine phosphorylation of IR and IRS-1 and increased basal phosphorylation of IRS-2, Akt, and mTOR when compared to controls. Basal glucose uptake was increased in adipocytes from UN-group and the treatment with LY294002, induced only a partial inhibition both in basal and in insulin-stimulated glucose uptake, suggesting an involvement of PI3K activity. These alterations were accompanied by higher GLUT-4 content in the plasma membrane and alterations in the actin cytoskeleton dynamics. These data suggest that early postnatal undernutrition impairs insulin sensitivity in adulthood by promoting changes in critical steps of insulin signaling in adipose tissue, which may contribute to permanent changes in glucose homeostasis.