p62^dok and Dok-3 are members of the Dok family of adaptors found in B cells, with the former cloned as a substrate of the p210^bcr/abl oncoprotein in Ph + chronic myelogenous leukemia. A role for p62^dok in FcγRIIB–mediated negative regulation of B-cell proliferation had been established previously. Here, we generated Dok-3^−/− mice to assess the function of Dok-3 in B cells. Mice lacking Dok-3 have normal B-cell development but possess higher level of IgM antibodies in their sera. In comparison to wild-type mice, Dok-3^−/− mice mounted significantly enhanced humoral immune responses to T cell–independent type I and II antigens. Dok-3–deficient B cells hyperproliferated, exhibited elevated level of calcium signaling as well as enhanced activation of NF-κB, JNK, and p38MAPK in response to B-cell receptor (BCR) engagement. In the absence of Dok-3, the localization of the inhibitory phosphatase SHIP-1 to the plasma membrane is intact while its phosphorylation is compromised, suggesting that Dok-3 could function to facilitate or sustain the activation of SHIP-1. The phenotype and responses of Dok-3^−/− mice and B cells could be differentiated from those of the Dok-1^−/− counterparts. Hence, we propose that Dok-3 plays a distinct and nonredundant role in the negative regulation of BCR signaling.