Engagement of the BCR with Ags triggers signaling pathways for commitment of B lymphocyte responses that can be regulated, in part, by reactive oxygen species. To investigate the functional relevance of reactive oxygen species produced in primary B cells, we focused on the role of the hydrogen peroxide generator Duox1 in stimulated splenic B cells under the influence of the TH2 cytokine IL-4. We found that H2O2 production in wild type (WT) and Nox2-deficient CD19+ B cells was boosted concomitantly with enhanced expression of Duox1 following costimulation with BCR agonists together with IL-4, whereas stimulated Duox1-/- cells showed attenuated H2O2 release. We examined whether Duox1-derived H2O2 contributes to proliferative activity and Ig isotype production in CD19+ cells upon BCR stimulation. Duox1-/- CD19+ B cells showed normal responses of Ig production but a higher rate of proliferation than WT or Nox2-deficient cells. Furthermore, we demonstrated that the H2O2 scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of WT CD19+ B cells in vitro. Results from immunized mice reflected the in vitro observations: T cell-independent Ag induced increased B cell expansion in germinal centers from Duox1-/- mice relative to WT and Nox2-/- mice, whereas immunization with T cell-dependent or -independent Ag elicited normal Ig isotype secretion in the Duox1 mutant mice. These observations, obtained both by in vitro and in vivo approaches, strongly suggest that Duox1-derived hydrogen peroxide negatively regulates proliferative activity but not Ig isotype production in primary splenic CD19+ B cells.