Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit β-arrestin and induce neutrophil chemotaxis, supporting the previous notion that β-arrestin translocation is of importance for cell migration. Despite the lack of β-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit β-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca2+, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of β-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.,Copyright ? 2019 by The American Association of Immunologists, Inc.