To clarify the roles of insulin on the proliferation and differentiation of intestinal epithelial cells (IECs), we examined the effect of insulin-like growth factor-I (IGF-I) and insulin for the growth and differentiation of IEC-6 cells, a crypt cell line derived from rat small intestine. IGF-I (100 nM) stimulated the proliferation of IEC-6 cells, and insulin (1-100 nM) antagonized the IGF-I effect and caused the cells' G1-arrest, resulting in differentiated characteristics of IECs, such as increased general protein synthesis and the formation of microvilli. To clarify the mechanisms of these phenomena, cell surface [125I]insulin binding and the content of immunoreactive insulin receptors were analyzed by Western blotting. Insulin receptors transiently appeared on the cell surface during the early G1 phase after the IGF-I stimulation. Under those conditions, the concomitant presence of insulin stimulated the appearance of active transforming growth factor-beta1 (TGF-beta1) in the media, and then TGF-beta1 antagonized the IGF-I-induced cell proliferation. Such a TGF-beta1 effect was blunted by a neutralizing antibody against TGF-beta1, indicating that the insulin effect was in part mediated through the autocrine-paracrine secretion of TGF-beta1. These results suggest that the regulation of the proliferation of IECs are an early step in those cells' differentiation that may accompany hormonal changes during nutrient intake and may be caused by the sequential effects of IGF-I, insulin, and TGF-beta1.