Mast cells and basophils express the high affinity receptor for IgE (FcepsilonRI) and play a central role for IgE-associated immediate hypersensitivity reactions and allergic disorders. Cross-linking of FcepsilonRI-bound IgE with multivalent antigen initiates the activation of mast cells and basophils, resulting in the degranulation from these cells. We have recently identified a novel inhibitory receptor, myeloid-associated immunoglobulin-like receptor (MAIR)-I, which is expressed on mast cells as well as other myeloid cell lineages. Co-ligation of FcepsilonRI and MAIR-I inhibits IgE-mediated degranulation from mast cells. However, MAIR-I-mediated signaling pathways involved in the inhibition remain undetermined. Here, we demonstrate that the transfectant of rat basophil leukemia RBL-2H3 expressing wild-type MAIR-I is tyrosine phosphorylated and recruits SHP-1 and SHIP upon cross-linking of MAIR-I. By using RBL-2H3 transfectants expressing variable mutant MAIR-I at Y233, Y258, Y270 and/or Y299, we further demonstrate that both Y258 and Y270, but not Y233 and Y299, were phosphorylated and were essentially required for inhibition of IgE-mediated degranulation from the RBL-2H3 transfectant.