CD81 is a component of the CD19/CD21 co-receptor complex in B cells. However, the role of CD81 in B cell activation has not been clearly elucidated. Here, we demonstrate that Cd81^−/− B cells stimulated via their B cell receptor fluxed higher intracellular-free calcium ion along with increased phosphorylation of spleen tyrosine kinase and phospholipase gamma 2. Additionally, Cd81^−/− B cells responded to toll like receptor 4 stimulation with increased nuclear factor-kappa B activation, cell proliferation and antibody secretion compared with wild-type B cells. Cd81^−/− mice also mounted a significantly higher immune response to T-independent antigens than their wild-type counterparts. Finally, analysis of Cd81^−/− B cells that were generated by bone marrow transplantation into Rag1^−/− mice confirmed that the hyperactive phenotype is not dependent on the CD81-deficient environment. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.