Staphylococcal alpha-toxin is an important virulence factor for Staphylococcus aureus to cause severe infections. In this study, we explored whether the toxoid of alpha-toxin may be utilized to block the toxicity of wild-type alpha-toxin. We created a series of H35A mutated alpha-toxin expression strains and revealed that the H35A mutation eliminates the activity of alpha-toxin using a human lung epithelial cell line (A549). More importantly, we found that either the pretreatment or simultaneous treatment of the epithelial cells with alpha-toxin-H35A completely disrupted the cytotoxicity of alpha-toxin. Specifically, we demonstrated that alpha-toxin-H35A can effectively interfere with the pore formation and the internalization of alpha-toxin using cytotoxicity and immunofluorescence assays. In addition, we found that the removal of either the 30-amino-acid (aa) or 99-aa C-terminal region of alpha-toxin-H35A reactivated its cytotoxicity, indicating that interactions between the alanine residue at position 35 and these C-terminal regions may be associated with interrupting the toxic activity of alpha-toxin-H35A. Taken together, these results suggest that the alpha-toxin-H35A protein may be developed as a potential alternative therapeutic agent for treating early stages of S. aureus infections.