We have investigated whether the mechanism by which the non-anticoagulant N-acetyl-de-O-sulfated-heparin (NSH) inhibits leukocyte infiltration is mediated by an effect on platelet function. We show that oral treatment with two doses of NSH significantly inhibits eosinophil and neutrophil recruitment into the lungs. Intravital microscopy analysis shows that NSH inhibits leukocyte and platelet diapedesis in the microcirculation of the cremaster muscle and in the trachea. More importantly, there were significantly lower numbers of leukocytes recruited into the lung in response to LPS in thrombocytopenic mice when transfused with platelets pretreated with NSH in vitro when compared with mice transfused with untreated platelets. Using intravital analysis of the microvasculature of the cremaster muscle, we have demonstrated that the reinfusion of activated platelets significantly re-established leukocyte diapedesis in response to LPS but that this effect was not observed when platelets were pretreated in vitro with NSH. Finally, we investigated whether the effect of NSH altered the expression of adhesion molecules on the surface of platelets and leukocytes in blood samples collected from mice treated orally with NSH. Our results demonstrate that NSH significantly inhibited the detection of P-selectin as evaluated by flow cytometry, confirming that part of the antiinflammatory action of NSH is via an effect on platelet function.