Vitamin D binding protein (DBP), also called Gc globulin, is an abundant plasma protein belonging to the albumin gene family. DBP is primarily known for its role in the transport of vitamin D metabolites in the blood and extracellular fluids, and as an actin scavenger in the circulation. DBP also has been shown to be a chemotactic cofactor for the complement activation peptide C5a, and a deglycosylated form (DBP-MAF) has been shown to activate macrophages and osteoclasts in vitro. The in vivo relevance and the mechanism behind the chemotactic cofactor function of DBP is not known. In this report, a C5a induced alveolitis model was developed and the neutrophil migration into the lungs of DBP -/- and DBP +/+ mice was determined. There was a significant decrease in the number of neutrophils recruited into the lung at 4, 6, and 24 hours after instillation of C5a in DBP -/- mice as compared to DBP +/+ animals. Ex vivo analysis of DBP -/- bone marrow neutrophils revealed decreased surface expression CD88 (C5a receptor), CD44 (DBP receptor), CD11b (cell adhesion molecule) and decreased side scatter (less internal granularity). Functional analysis of DBP -/- bone marrow neutrophils revealed an intrinsic defect in migration, polarization and superoxide anion generation suggesting a role for DBP in neutrophil differentiation in vivo. This was confirmed using the promyelocytic cell line HL-60, where higher surface expression of CD88, CD44 and higher side scatter was observed when cells were differentiated in the presence of DBP. Finally, a mechanism by which DBP functions during chemotaxis and differentiation is proposed. Using a proteomic approach, it was observed that DBP treated neutrophils generate DBP-actin complexes which in turn induce secretion the inflammatory amplifier S100A8/A9. This molecule has been implicated in neutrophil chemotaxis, polarization, superoxide anion generation and differentiation. In vitro analysis of human neutrophils confirmed that DBP treatment induced the generation of DBP-actin complexes and the formation of intracellular S100A8/A9 complexes. Furthermore, DBP-actin complexes were found to be potent inducers of S100A8/A9 release from neutrophils.