CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4+ T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.