Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A₂ (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including <i>S. aureus</i>. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of <i>lspA</i>, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the <i>lspA</i> mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, <i>lspA</i> deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of &#x3e;26,000 <i>S. aureus</i> genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since <i>Streptococcus mutans</i> and <i>Enterococcus faecalis</i> were also more hGIIA-susceptible after <i>lspA</i> deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.