Destruction of pancreatic islet β-cells in type 1 diabetes (T1D) is mainly mediated by autoimmune T and B lymphocytes. We reported that induction of major histocompatibility complex (MHC)–mismatched mixed chimerism reversed autoimmunity and reestablished thymic negative selection of autoreactive T cells in NOD mice, but it is still unclear how mixed chimerism tolerizes autoreactive B cells. The current studies were designed to reveal the mechanisms on how mixed chimerism tolerizes autoreactive B cells in T1D. Accordingly, mixed chimerism was induced in NOD mice through radiation-free nonmyeloablative anti-CD3/CD8 conditioning and infusion of donor CD4+ T cell–depleted spleen and whole bone marrow (BM) cells or through myeloablative total body irradiation conditioning and reconstitution with T cell–depleted BM cells from donor and host. Kinetic analysis of percentage and yield of preplasma and plasma B cells, newly developed B-cell subsets, and their apoptosis was performed 30–60 days after transplantation. Induction of MHC-mismatched mixed chimerism results in depleting host-type pre-existing preplasma and plasma B cells as well as augmenting apoptosis of immature transitional T1 B cells, including insulin-specific B cells in a donor B cell–dependent manner. Therefore, induction of MHC-mismatched mixed chimerism depletes pre-existing and de novo–developed autoreactive B cells.