Chemotactic migration of neutrophils to peripheral tissues is a protective immune response.Yet, neutrophil accumulation often mediates tissue injury. Since neutrophil migration depends on protrusion of a lamellipodium and cytoskeletal reorganization, we asked whether a synthetic peptide conjugate known to stabilize fibroblast actin filaments and cell shape would modulate neutrophil chemotaxis. The peptide of multivalent P34BSA is identical to a 10-amino acid sequence in Treponema denticola major outer sheath protein (Msp). Chemotaxis was studied in mice using a dermal air pouch model and an acute peritonitis model. Local injection of P34BSA prior to lipopolysaccharide or Zymosan A, respectively, greatly diminished the accumulation of neutrophils (hemocytometer count) and myeloperoxidase activity (enzyme assay) in fluid aspirates, compared with P97BSA or P96BSA, control conjugates with either a scrambled sequence or with arginine residues substituted for lysines. Histology of the dermal pouch tissues found that the control-pretreated pouches were greatly distended, whereas the P34BSA-pretreated pouch was narrow, as if collapsed. P34BSA pretreatment of neutrophils in vitro inhibited formyl-Methionine-Leucine-Phenylalanine (fMLP)-activated cell polarization and chemotactic migration in Zigmond chambers. fMLP-activation of Rho family small GTPases was measured with commercial ELISA kits. Pretreatment with P34BSA boosted RhoA activation, inhibited Rac1 activation, and had no effect on Cdc42. Peptide conjugate P34BSA inhibits neutrophil migration in experimental acute migration assays in mice and in vitro, and it alters fMLP activation of GTPases known to regulate neutrophil polarization.