Antimicrobial peptides produced by epithelial and immune cells protect tissues from various infections. Whether enterovirus infection leads to stimulation of antimicrobial peptide expression is unknown. We examined antimicrobial peptide mRNA and protein production in HT-29 colon adenocarcinoma cells infected with picornaviruses. The antiviral activity of increased antimicrobial peptide production was evaluated by using a recombinant peptide and corresponding gene overexpression. Enterovirus infection enhanced both the mRNA expression and secretion of human β-defensin (hBD) 3 in intestinal epithelial cells but did not increase expression of human neutrophil peptide 1-3 (HNP 1-3), HNP4, human defensin 5 (HD5), HD6, hBD1, hBD2, and hBD5. The recombinant but not the intracellularly overexpressed hBD3 inhibited enterovirus (EV) 71, coxsackievirus A16 (CVA16), CVB5 and poliovirus 1 (PV1) infecting HT-29 cells. Our results suggest that enterovirus infection induces hBD3 production in human intestinal epithelial cells and that hBD3 can exert extracellular anti-enterovirus activity.