Human Respiratory Syncytial Virus (HRSV) is an important pathogen and is associated with mortality in the young, old, and immuno-compromised. Due to the lack of effective therapeutic antivirals or a vaccine, there is a critical need for continued research in this field. Here we tested the ability of the FDA approved proteasome inhibitor bortezomib to inhibit HRSV in vitro and in vivo. We observed significant inhibition of HRSV replication in Vero cells at bortezomib concentrations from 20 to 40 ng/ml. Bortezomib was well tolerated in mice when administered intranasally at concentrations of < or = 0.3 mg/kg or intraperitoneally at 1.0 mg/kg. However, treatment of HRSV-infected mice with doses as low as 0.01 mg/kg resulted in increased pulmonary inflammation and mortality compared to mock treated-infected control animals. Examination of cytokine expression levels from lungs of bortezomib treated HRSV-infected mice revealed an increase in G-CSF, IL-6, MCP-1, and RANTES levels and a decrease in total IL-12 compared to mock treated-infected control animals. These data indicate that treatment with bortezomib during HRSV infection may alter the immune response and could potentially create a risk for patients treated with bortezomib in the event of a respiratory tract infection.