Human colon epithelial cells express the G protein-coupled receptor CCR6, the sole receptor for the chemokine CCL20 (also termed MIP-3alpha). CCL20 produced by intestinal epithelial cells is upregulated in response to proinflammatory stimuli and microbial infection, and it chemoattracts leukocytes, including CCR6-expressing immature myeloid dendritic cells, into sites of inflammation. The aim of this study was to determine whether CCR6 expressed by intestinal epithelial cells acts as a functional receptor for CCL20 and whether stimulation with CCL20 alters intestinal epithelial cell functions. The human colon epithelial cell lines T84, Caco-2, HT-29, and HCA-7 were used to model colonic epithelium. Polarized intestinal epithelial cells constitutively expressed CCR6, predominantly on the apical side. Consistent with this, apical stimulation of polarized intestinal epithelial cells resulted in tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), an adaptor/scaffolding protein that localizes in focal adhesions and has a role in regulating cytoskeletal elements important for cell attachment and migration. In addition, CCL20 stimulation inhibited agonist-stimulated production of the second messenger cAMP and cAMP-mediated chloride secretory responses by intestinal epithelial cells. Inhibition was abrogated by pertussis toxin, consistent with signaling through Galphai proteins that negatively regulate adenylyl cyclases and cAMP production. These data indicate that signaling events, occurring via the activation of the apically expressed chemokine receptor CCR6 on polarized intestinal epithelial cells, alter specialized intestinal epithelial cell functions, including electrogenic ion secretion and possibly epithelial cell adhesion and migration.