Phosphatidylinositol 3-kinase (PI3K) pathway of leptin signaling plays an important role in transducing leptin action in the hypothalamus. Obesity is usually associated with resistance to the effect of leptin on food intake and energy homeostasis. Although central leptin resistance is thought to be involved in the development of diet-induced obesity (DIO), the mechanism behind this phenomenon is not clearly understood. To determine whether DIO impairs the effect of leptin on hypothalamic PI3K signaling, we fed 4-wk-old FVB/N mice a high-fat diet (HFD) or low-fat diet (LFD) for 19 wk. HFD-fed mice developed DIO in association with hyperleptinemia, hyperinsulinemia, and impaired glucose and insulin tolerance. Leptin (ip) significantly increased hypothalamic PI3K activity and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) levels in LFD-fed mice but not in DIO mice. Immunocytochemical study confirmed impaired p-STAT3 activation in various hypothalamic areas, including the arcuate nucleus. We next tested whether both PI3K and STAT3 pathways of leptin signaling were impaired during the early period of DIO. Leptin failed to increase PI3K activity in DIO mice that were on a HFD for 4 wk. However, leptin-induced p-STAT3 activation in the hypothalamus measured by Western blotting and immunocytochemistry remained comparable between LFD- and HFD-fed mice. These results suggest that the PI3K pathway but not the STAT3 pathway of leptin signaling is impaired during the development of DIO in FVB/N mice. Thus, a defective PI3K pathway of leptin signaling in the hypothalamus may be one of the mechanisms of central leptin resistance and DIO.