The mechanism by which insulin induces the expression of the sterol regulatory element binding protein 1c (SREBP-1c) and glucokinase genes was investigated in cultured rat hepatocytes. Overexpression of an NH₂-terminal fragment of IRS-1 that contains the pleckstrin homology and phosphotyrosine binding domains (insulin receptor substrate-1 NH₂-terminal fragment [IRS-1N]) inhibited insulin-induced tyrosine phosphorylation of IRS-1 as well as the association of IRS-1 with phosphatidylinositol (PI) 3-kinase activity, whereas the tyrosine phosphorylation of IRS-2 and its association with PI 3-kinase activity were slightly enhanced. The equivalent fragment of IRS-2 (IRS-2N) prevented insulin-induced tyrosine phosphorylation of both IRS-1 and IRS-2, although that of IRS-1 was inhibited more efficiently. The insulin-induced increases in the abundance of SREBP-1c and glucokinase mRNAs, both of which were sensitive to a dominant-negative mutant of PI 3-kinase, were blocked in cells in which the insulin-induced tyrosine phosphorylation of IRS-1 was inhibited by IRS-1N or IRS-2N. A dominant-negative mutant of Akt enhanced insulin-induced tyrosine phosphorylation of IRS-1 (but not that of IRS-2) and its association with PI 3-kinase activity, suggesting that Akt contributes to negative feedback regulation of IRS-1. The Akt mutant also promoted the effects of insulin on the accumulation of SREBP-1c and glucokinase mRNAs. These results suggest that the IRS-1–PI 3-kinase pathway is essential for insulin-induced expression of SREBP-1c and glucokinase genes.