The opportunistic gram-positive pathogen Staphylococcus aureus is a leading cause of pneumonia and sepsis. Staphylococcal a-toxin, a prototypical pore-forming toxin, is a major virulence factor of S. aureus clinical isolates, and lung epithelial cells are highly sensitive to a-toxin's cytolytic activity. Type I interferon (IFN) signaling activated in response to S. aureus increases pulmonary cell resistance to a-toxin, but the underlying mechanisms are uncharacterized. We show that IFNa protects human lung epithelial cells from a-toxin-induced intracellular ATP depletion and cell death by reducing extracellular ATP leakage. This effect depends on protein palmitoylation and induction of phospholipid scramblase 1 (PLSCR1). IFNa-induced PLSCR1 associates with the cytoskeleton after exposure to a-toxin, and cellular depletion of PLSCR1 negates IFN-induced protection from a-toxin. PLSCR1-deficient mice display enhanced sensitivity to inhaled a-toxin and an a-toxin-producing S. aureus strain. These results uncover PLSCR1 activity as part of an innate protective mechanism to a bacterial pore-forming toxin.