Parainfluenza viruses are a common cause of seasonal respiratory disease, but in high-risk individuals (e.g., young children) these viruses can cause severe clinical manifestations that require hospitalization. Beta-defensins are a subclass of antimicrobial peptides with antiviral activity. Use of adenovirus-mediated beta-defensin gene expression has been proposed as therapy for chronic bacterial infections commonly seen in cystic fibrosis patients; however, its use during parainfluenza virus 3 (PIV3) infection has not been evaluated. The hypothesis in this experiment was that adenovirus expression of human beta-defensin 6 (HBD6) would diminish concurrent PIV3 infection in neonatal lambs. The group infected with adenovirus HBD6 and PIV3 had increased levels of pulmonary neutrophil recruitment compared to those for the group infected with PIV3 or PIV3 and adenovirus, with an increased respiration rate and body temperature late in the course of the PIV3-adenovirus HBD6 infection. Interestingly, the adenovirus-treated groups had higher levels of immunohistochemical staining for PIV3 and syncytial cell formation than the group infected with PIV3, suggesting that treatment with the adenovirus vector, regardless of whether it was carrying a target gene, exacerbated the PIV3 infection. The levels of expression of mRNA for antimicrobial surfactant proteins A and D and sheep beta-defensin 1 were increased by PIV3 and adenovirus treatment, and the increased levels of expression roughly corresponded to the degree of inflammation. While pulmonary administration of a high-dose adenovirus vector has been associated with undesirable inflammation, this is the first study to show that it can exacerbate concurrent viral infection, a concern that needs to be addressed for future studies of adenovirus in the lung. Additionally, this study showed that adenovirus-mediated HBD6 expression increases neutrophil recruitment, a recently described attribute of beta-defensins, with mild accentuation of PIV3 activity and inflammation.