Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF)-alpha, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-alpha, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.