The present study examined the effect of 5-hydroxytraptamine (5-HT) on the feeding behavior of transgenic mice with neuropeptide Y (NPY) overexpression. Solution of 5-HT (1, 2.5 or 5 mg/kg) was administered intraperitoneally into (1) NPY-overexpressing mice, and (2) wild-type mice with 2-deoxy-d-glucose (2-DG) induced hyperphagia. The NPY-overexpressing mice were further divided into five groups: (1) control mice, (2) mice treated with 5-HT (5 mg/kg), (3) mice treated with 5-HT (5 mg/kg) and ketanserin (0.5 or 1 mg/kg), a 5-HT2A receptor antagonist, (4) mice treated with insulin (1 IU/kg), and (5) mice treated with insulin (1 IU/kg) and 5-HT (5 mg/kg). Food intake and plasma glucose levels were measured. The results showed that 5-HT reduced hyperphagia in both NPY-overexpressing mice and 2-DG-treated mice in dose-dependent manner. Hyperglycemia was induced by 5-HT administration. Ketanserin antagonized the 5-HT induced hypophagia and hyperglycemia. Insulin, on the other hand, prevented 5-HT induced hyperglycemia but not the hypophagic effect. In conclusion, 5-HT reduces hyperphagia in the NPY-overexpressing rat through action on 5-HT2A receptors and this hypophagic effect of 5-HT does not depend on the hyperglycemia.