Adaptor protein 3BP2 positively regulates the high affinity IgE receptor (FcɛRI)‐mediated activation of degranulation in mast cells. Genetic study identified the point mutations of 3BP2 gene in human‐inherited disease cherubism. The multiple cysts in cherubism lesion of jaw bones are filled with the activated osteoclasts and stromal cells, including mast cells. By over‐expression study using rat basophilic leukaemia RBL‐2H3 mast cells, we have analysed the effect of the point mutations on the function of 3BP2 protein, which plays a positive regulatory role on FcɛRI‐mediated mast cell activation. Over‐expression of 3BP2 mutants suppressed the antigen‐induced degranulation and cytokine gene transcription. Antigen‐induced phosphorylation of Vav1, activation of Rac1, extracellular signal regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), inhibitor of nuclear factor κB kinase (IKK) and nuclear factor of activated T cells (NFAT) were all impaired in the cells over‐expressing the cherubism mutants of 3BP2. Furthermore, cherubism mutations of 3BP2 may abrogate the binding ability to interact with chaperone protein 14‐3‐3. These results demonstrate that over‐expression of the mutant form of 3BP2 inhibits the antigen‐induced mast cell activation. It suggests that point mutations of 3BP2 gene cause the dysfunction of 3BP2 in vivo.