The major outer sheath protein (Msp), a virulence determinant of the periodontal pathogen Treponema denticola, is known to perturb calcium and cytoskeletal dynamics that impacts cellular migration of fibroblasts. The aim of this study was to investigate the effects of enriched native Msp on key antimicrobial functions of human neutrophils (PMNs), namely chemotaxis, phagocytosis, and oxidative responses to agonists. Pretreatment of PMNs with Msp led to inhibition of chemotaxis and phagocytosis. Additionally, pretreatment of PMNs with Msp diminished fMLP-stimulated (i) amplitude of cytosolic Ca2+ transients, and (ii) the number of actin filament free barbed ends. However, Msp had no effect on PMN oxidative responses to fMLP or to phorbol myristate acetate, measured by the oxidation of dihydrorhodamine 123 by flow cytometry, and it did not induce apoptosis or plasma membrane permeability. T. denticola Msp selectively impairs human neutrophil locomotion, chemotaxis and phagocytosis in vitro through its impact on the cytoskeleton.