Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis.