Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted. Copyright © 2009, American Society for Microbiology. All Rights Reserved.