Previous studies have demonstrated the existence of a subset of B lymphocytes, regulatory B cells (Bregs), which modulate immune function. In this study, in vivo and in vitro experiments were undertaken to elucidate the role of these Bregs in controlling neuroinflammation following viral brain infection. We used multicolor flow cytometry to phenotype lymphocyte subpopulations infiltrating the brain, along with in vitro cocultures to assess their anti-inflammatory and immunoregulatory roles. This distinctive subset of CD19+CD1dhiCD5+ B cells was found to infiltrate the brains of chronically infected animals, reaching highest levels at the latest time point tested (30 d postinfection). B cell-deficient Jh-/- mice were found to develop exacerbated neuroimmune responses as measured by enhanced accumulation and/or retention of CD8+ T cells within the brain, as well as increased levels of microglial activation (MHC class II). Conversely, levels of Foxp3+ regulatory T cells were found to be significantly lower in Jh-/- mice when compared with wild-type (Wt) animals. Further experiments showed that in vitro-generated IL-10-secreting Bregs (B10) were able to inhibit cytokine responses from microglia following stimulation with viral Ags. These in vitro-generated B10 cells were also found to promote proliferation of regulatory T cells in coculture studies. Finally, gain-of-function experiments demonstrated that reconstitution of Wt B cells into Jh-/- mice restored neuroimmune responses to levels exhibited by infected Wt mice. Taken together, these results demonstrate that Bregs modulate T lymphocyte as well as microglial cell responses within the infected brain and promote CD4+Foxp3+ T cell proliferation in vitro.