Intracerebroventricular injection and overexpression of Neuropeptide Y (NPY) in the paraventricular nucleus (PVN) has been shown to induce obesity and glucose metabolism disorder in rodents; however, the underlying mechanisms are still unclear. The aim of this study was to investigate the mechanism contributing to glucose metabolic disturbance induced by NPY. Recombinant lentiviral NPY vectors were injected into the PVN of rats fed a high fat (HFD) or low-fat diet. 8 weeks later, in vivo intravenous glucose tolerance tests and euglycemic-hyperinsulinemic clamp revealed that insulin resistance of adipose tissue were induced by NPY overexpression with or without HFD. NPY increased food intake, but did not change blood glucose, glycated hemoglobin A1c (HbA1c) or lipid levels. However, NPY decreased the expression of pGSK3β, PI3K p85 and pAKTSer473 in adipose tissue of rats. In vitro, 3T3-L1 adipocytes were treated with NPY, NPY Y1 and Y5 receptor antagonists. Glucose consumption and 2-deoxy-D-[3H] glucose uptake were partly inhibited by NPY, while a decrease in PI3K-AKT pathway signaling and a decreased expression of pGSK3α and pGSK3β were observed. Nevertheless, a Y5 receptor antagonist (L-152,804) reversed the effects of NPY on glucose uptake and consumption. These data suggest that long-term over-expression of NPY in PVN contributes to the establishment of adipose tissue insulin resistance, at least partly via the Y5 Receptor.