PTEN (phosphatase and tensin homolog) dephosphorylates phosphatidylinositol 3,4,5-triphosphate and antagonizes PI 3-kinase. Insulin acts in the mediobasal hypothalamus (MBH) not only to suppress food intake and weight gain, but also to improve glucose metabolism, via PI 3-kinase activation. Thus, blocking hypothalamic PTEN is a potential target for treating obesity as well as diabetes. However, genetic modification of PTEN in specific neuronal populations in the MBH yielded complex results, and no postnatal intervention for hypothalamic PTEN has yet been reported. In order to elucidate how postnatal modification of hypothalamic PTEN influences food intake as well as glucose metabolism, we bidirectionally altered PTEN activity in the MBH of rats by adenoviral gene delivery. Inhibition of MBH PTEN activity reduced food intake and weight gain, while constitutive activation of PTEN tended to induce the opposite effects. Interestingly, the effects of MBH PTEN intervention on food intake and body weight were blunted by high-fat feeding. However, MBH PTEN blockade improved hepatic insulin sensitivity even under high-fat fed conditions. On the other hand, constitutive activation of MBH PTEN induced hepatic insulin resistance. Hepatic Akt phosphorylation and the G6Pase expression level were bidirectionally modulated by MBH PTEN intervention. These results demonstrate that PTEN in the MBH regulates hepatic insulin sensitivity, independently of the effects on food intake and weight gain. Therefore, hypothalamic PTEN is a promising target for treating insulin resistance even in states of over-nutrition.