Objectives: B cells are important in the pathogenesis of multiple sclerosis. It is yet unknown which subsets may be involved, but atypical B cells have been proposed as mediators of autoimmunity. In this study, we investigated differences in B-cell subsets between controls and patients with untreated and anti-CD20-treated multiple sclerosis. Methods: We recruited 155 participants for an exploratory cohort comprising peripheral blood and cerebrospinal fluid, and a validation cohort comprising peripheral blood. Flow cytometry was used to characterize B-cell phenotypes and effector functions of CD11c⁺ atypical B cells. Results: There were no differences in circulating B cells between controls and untreated multiple sclerosis. As expected, anti-CD20-treated patients had a markedly lower B-cell count. Of B cells remaining after treatment, we observed higher proportions of CD11c⁺ B cells and plasmablasts. CD11c⁺ B cells were expanded in cerebrospinal fluid compared to peripheral blood in controls and untreated multiple sclerosis. Surprisingly, the proportion of CD11c⁺ cerebrospinal fluid B cells was higher in controls and after anti-CD20 therapy than in untreated multiple sclerosis. Apart from the presence of plasmablasts, the cerebrospinal fluid B-cell composition after anti-CD20 therapy resembled that of controls. CD11c⁺ B cells demonstrated a high potential for both proinflammatory and regulatory cytokine production. Interpretation: The study demonstrates that CD11c⁺ B cells and plasmablasts are less efficiently depleted by anti-CD20 therapy, and that CD11c⁺ B cells comprise a phenotypically and functionally distinct, albeit heterogenous, B-cell subset with the capacity of exerting both proinflammatory and regulatory functions.