Although defensins are known to inhibit the replication of human influenza A virus (IAV) in vitro, their in vivo expression during IAV infection is not known. Here we investigated mRNA and protein expression of several beta-defensins in the airways of IAV infected mice. Expression of murine beta-defensin (MBD)-3 and -4 was enhanced (3 to 5-fold, p<0.01) in infected lungs, trachea and sinonasal mucosa. MBD-3 and -4 expressions were correlated with the time course of acute IAV infection suggesting their induction by IAV infection. Infected mice also showed increased levels of surfactant protein-D especially in the sinonasal mucosa. MBD-3 and -4 were localized to the conducting airway epithelial cells but not the alveolar tissue. We conclude that IAV infection upregulated the expression of inducible beta-defensins in both the upper and lower airways. These novel findings suggest that beta-defensins might contribute to innate and adaptive immune responses targeted against IAV infection.