AbstractLimiting microbial threats, maintenance and re‐establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human β‐defensin 2 (hBD‐2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco‐2 and HT‐29 cells were stimulated with 0.1–10 µg/ml hBD‐2 for 6–72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo‐deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot‐blot analysis. Furthermore, anti‐apoptotic capacities of hBD‐2 were studied. Over a broad range of concentrations and stimulation periods, hBD‐2 was well tolerated by IECs and did not induce apoptosis. hBD‐2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD‐2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF‐related apoptosis‐inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD‐2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD‐2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease. J. Cell. Biochem. 104: 2286–2297, 2008. © 2008 Wiley‐Liss, Inc.