In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.