Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-β (TGF-β). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-β-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-β-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-β-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70 S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-β-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70 S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-β was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-β-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-β-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.