Insulin action in the hypothalamus produces sympathetic nerve activation with pathological implications in obesity and type 2 diabetes. However, the molecular events mediating the sympathetic effects evoked by hypothalamic action of insulin are poorly understood. Mammalian target of rapamycin (mTOR) which can be activated by the insulin receptor has emerged as an important regulator of sympathetic nerve activity (SNA). Phosphatidylinositol 3-kinase (PI3K) pathway is an important upstream regulator of mTOR signaling; and insulin stimulates PI3K in the hypothalamus. Thus, we postulate that the PI3K-mTOR axis is critical for insulin-induced sympathetic nerve activation. In hypothalamic GT1-7 neurons, we found that insulin (100 nM) activated mTOR signaling as indicated by the increase in phosphorylated levels of the downstream effectors, S6K (1.5-fold) and S6 (1.4-fold), measured by Western blot. In GT1-7 neurons, insulin also increased the phosphorylation levels of Akt (2.7-fold), a downstream effector of PI3K. Importantly, inhibition of PI3K (LY294202, 10 μM) abolished insulin-induced activation of S6K and S6. To study further the contribution of PI3K signaling to the insulin action, we used p110αD933/WT mice which carry a heterozygous mutation disrupting PI3K signaling. In wild type (WT) control mice, ICV administration of insulin (100 μU) significantly elevated the number of phospho-S6 positive cells in the hypothalamic arcuate nucleus (29±1, n=3, p<0.05 vs. vehicle), assessed by immunohistochemistry. This response was significantly attenuated in p110αD933/WT mice (12±3, n=3, p<0.05 vs. WT mice). Moreover, the increase in renal SNA, measured directly using multifiber recording, induced by ICV insulin (100 μU) was blunted (p<0.05) in the p110αD933A/WT mice (-15±25%, n=6) as compared to littermate controls (327±115%, n=6). In WT mice, ICV pre-treatment with an mTOR inhibitor (rapamycin, 1 ng) also attenuated the renal SNA response induced by ICV insulin (-16±14%, n=6), mimicking the effect of PI3K inhibition. These results suggest that hypothalamic PI3K-mTOR axis is a critical node in the control of renal sympathetic outflow by insulin.