Tyrosine phosphorylation of adaptor protein c‐Abl‐Src homology 3 (SH3) domain‐binding protein‐2 (3BP2, also referred to SH3BP2) positively regulates the B‐cell antigen receptor (BCR)‐mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B‐cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2‐P416R, corresponding to P418R in human protein) enhanced BCR‐mediated activation of NFAT. 3BP2‐P416R increased the signaling complex formation with Syk, phospholipase C‐γ2 (PLC‐γ2), and Vav1. In contrast, 3BP2‐P416R could not change the association with the negative regulator 14‐3‐3. Loss of the association mutant that was incapable to associate with 14‐3‐3 could not mimic BCR‐mediated NFAT activation in Syk‐deficient cells. Moreover, BCR‐mediated phosphorylation of extracellular signal regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules.