Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case-fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, it was cloned and expressed in human airway epithelial cells (NCI-H292). The apoptotic events at post-transfection were examined by the terminal transferase dUTP nick end labeling (TUNEL) assay, flow cytometric measurement of propidium iodide (PI), annexin V staining, and Western blot analyses with antibodies specific for pro-apoptotic and anti-apoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase (PARP) and activation of caspases-3, -7, -8 during the NS1-induced apoptosis. Caspase-inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1-NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (time to >30% apoptosis was reduced from 24 hr to 6 hr post-transfection). Furthermore, after 24 hr post-transfection, there was about a 5.6-fold increase in Fas ligand mRNA expression detected in H5N1-NS1 transfected cells. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.