Staphylococcus aureus causes suppurative infections which are often associated with tissue destruction and cell death. In the present study, we investigated the molecular and cellular basis of S. aureus-induced apoptosis and death in a human lung epithelial cell line (A549). We found that staphylococcal α-toxin is an important mediator of cytotoxicity in these epithelial cells. Specifically, we found that downregulating α-toxin production eliminated the cytotoxicity of S. aureus, whereas the addition of α-toxin to the cell culture medium significantly increased cell death in a dose-dependent manner. Importantly, we found that α-toxin-mediated cell death may partially function through α5β1-integrin, because both the β1-integrin antibody and the ligand fibronectin inhibited the cytotoxicity of α-toxin. Furthermore, we found that the overexpression of the inflammatory cytokine interferon (TNF)-α is associated with α-toxin-induced cell death, because both the TNF-α release inhibitor and antibody effectively inhibited the cytotoxicity of α-toxin. In contrast, the cytotoxicity of α-toxin was enhanced by the inhibition of the MAPK p38 and NF-κB pathways. Taken together, our results suggest that the activation of the MAPK p38 and NF-κB pathways are stress responses for survival, rather than direct contributes to α-toxin-induced cell death, and that the interaction of α-toxin with α5β1-integrin and overproduction of TNF-α may contribute to destruction of epithelial cells during S. aureus infection.