Staphylococcus aureus colonizes large segments of the human population and causes invasive infections due to its ability to escape phagocytic clearance. During infection, staphylococcal nuclease and adenosine synthase A convert neutrophil extracellular traps to deoxyadenosine (dAdo), which kills phagocytes. The mechanism whereby staphylococcal dAdo intoxicates phagocytes is not known. Here we used CRISPR-Cas9 mutagenesis to show that phagocyte intoxication involves uptake of dAdo via the human equilibrative nucleoside transporter 1, dAdo conversion to dAMP by deoxycytidine kinase and adenosine kinase, and signaling via subsequent dATP formation to activate caspase-3–induced cell death. Disruption of this signaling cascade confers resistance to dAdo-induced intoxication of phagocytes and may provide therapeutic opportunities for the treatment of infections caused by antibiotic-resistant S. aureus strains.