Autoreactive B cells are the source of pathogenic autoantibodies (autoAb) in systemic lupus erythematosus (SLE). Previous studies have demonstrated that anti-small nuclear ribonucleoprotein particles (snRNP) B cells from normal background mice tolerize T cells in the periphery and do not secrete autoAb. In this study, we examined whether these anti-snRNP B cells can be activated for autoAb production by the engagement of Toll-like receptors (TLR). Anti-snRNP B cells proliferated vigorously and secreted abundant anti-snRNP autoAb upon exposure to CpG or polyriboinosinic polyribocytidylic acid [poly (I:C)] in vitro. In addition, the costimulatory molecules CD80 and CD86 were up-regulated. While both anti-snRNP B cells and wild-type B cells produced similar levels of IL-6 and IL-10, anti-snRNP B cells secreted predominately IFN-gamma in response to CpG or poly (I:C) stimulation. Furthermore, we showed that in vivo engagement of TLR stimulated immature anti-snRNP B cells to further differentiate and produce autoAb and form germinal centers. The activated anti-snRNP B cells became expanded and migrated into the T-B cell interface. Moreover, TLR engagement directly or indirectly activated autoreactive B cells via a CD4 T cell-independent manner. These results provide in vitro and in vivo evidence that BCR/TLR co-engagement promotes the activation of anti-snRNP B cells for autoAb production.