Staphylococcus aureus is a well-known human bacterial pathogen and a leading cause of infective endocarditis (IE). Neutrophil migration, a common hallmark of IE, is a crucial host innate immune response that is prompted by localised bacterial infections. We have previously shown that LukAB, a pore-forming S. aureus leukotoxin, is significantly associated with S. aureus-related IE. Here, we examined the process of neutrophil chemotaxis in response to LukAB. To generate pure LukAB, the lukAB genes were cloned into an expression vector and LukAB were expressed and co-purified from Escherichia coli cells. The purity of recombinant LukAB was assessed by SDS-PAGE and the identify of LukA was confirmed with mass spectrometry. We utilized a Boyden Chamber assay, in which purified LukAB was placed in the bottom wells of a transwell plate, while human or murine neutrophils were isolated and seeded into the top wells. Migration of neutrophils to the bottom wells were quantified by a chromogenic substrate specific for neutrophil elastase. To examine the impact of LukAB on leukocyte recruitment in vivo, we utilised intravital microscopy to examine leukocyte-endothelial cell interaction in the mouse cremaster muscle (2). We found that purified LukAB significantly induced human and murine neutrophil chemotaxis in a concentration-dependent manner. These data were confirmed in vivo and in real-time using intravital microscopy of mouse cremaster muscles. Injection of expressed and purified LukAB into muscle tissue significantly induced leukocyte adherence (P<0.001) to blood vessels and leukocyte transmigration into surrounding tissues (P<0.001). LukAB directly induced leukocyte-endothelial interactions, with significant reductions in leukocyte rolling velocity (P < 0.05) and flux (P < 0.05) observed as early as 30 min after LukAB exposure. This study shows that in addition to being a leukotoxin, LukAB also acts as a direct neutrophil chemoattractant, likely potentiating its mechanism of action in IE and S. aureus pathogenesis.