Insulin receptor substrate (IRS) proteins are important intracellular molecules that mediate insulin receptor tyrosine kinase signaling. A decreased content of IRS proteins has been found in insulin-resistant states in animals, humans, and cultured cells under various conditions. However, the molecular mechanism that controls cellular levels of IRS proteins is unknown. We report that chronic insulin treatment induces the degradation of IRS-1, but not IRS-2, protein in cultured cells. The insulin-induced degradation of IRS-1 can be prevented by pretreatment with lactacystin, a specific inhibitor for proteasome degradation. These data demonstrate, for the first time, that insulin-induced degradation of IRS-1 is mediated by the proteasome degradation pathway. IRS-2 can escape from the insulin-induced proteasome degradation, suggesting the existence of specific structural requirements for this degradation process.