Neurotensin (NT), a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR)] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3) regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3alpha/beta in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3beta phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCalpha and PKCbeta1) or downregulation of endogenous PKCalpha or PKCbeta1 blocked NT-mediated GSK-3beta (but not GSK-3alpha) phosphorylation. Moreover, a selective PKCbeta inhibitor, LY379196, reduced NT-mediated GSK-3beta (but not GSK-3alpha) phosphorylation, suggesting a role for PKCbeta1 in the NT-mediated phosphorylation of GSK-3beta and an undefined kinase in the NT-mediated phosphorylation of GSK-3alpha. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.