In multiple sclerosis (MS), the immune damage to the central nervous system results from the net balance between self-reactive and immunoregulatory cells, among other factors. We identified novel perforin-expressing regulatory B-cells (BReg) in patients with clinical isolated syndrome, significantly enriched within the cerebrospinal fluid when compared to peripheral blood, of memory B cell phenotype (CD19+CD25+, CD19+CD25+FoxP3+and CD19+FoxP3+, p = 0.007, p = 0.06 and p = 0.03, respectively). These BRegsubsets were also higher in relapsing-remittent MS during relapse symptoms than in non-clinically active MS patients. Suppressive effects by CD19+CD25+ hi BReg on CD4+ T cells proliferation seem to be mediated at least in part by perforin/granzyme pathway. To our knowledge, this is the first report that shows cytolytic perforin/granzyme granules storage in B cells; the interesting point is its involvement on BRegcells immunosuppressive mechanisms, similarly to that in TRegcells. Our data may extend the understanding of pathophysiological processes in MS immunoregulation.