Previously, we demonstrated that macrophages from thrombospondin 1 (TSP1)-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In this study, we determined how TSP1 regulates macrophage function. We found that recombinant or purified platelet human TSP1 treatment stimulated tumor-necrosis factor (TNF)-α expression in bone marrow-derived macrophages in a time- and dose-dependent manner. Toll-like receptor 4 (TLR4) expression (at the mRNA and protein levels) and nuclear factor-kappaB (NF-κB) activity were also stimulated by TSP1 treatment. The TSP1-mediated increase in TNF-α production was abolished in TLR4-deficient macrophages, suggesting that TSP1 activates macrophages through a TLR4-dependent pathway. TSP1 also stimulated TLR4 activation in macrophages in vivo. Furthermore, TSP1-mediated macrophage activation was attenuated by using a peptide or an antibody to block the association between TSP1 and CD36. Taken together, these data suggest that the stimulation of the macrophage TLR4 pathway by TSP1 is partially mediated by the interaction of TSP1 with its receptor, CD36.