ABSTRACT Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM ⁺ mBc, we found that 62% and 21% of total (non-antigen-specific) IgM ⁺ and RV-IgM ⁺ mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM ⁺ (17%) and RV-IgM ⁺ (7%) mBc were lower than those of the corresponding switched (IgG ⁺ IgA ⁺ ) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM ⁺ RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ ^null ) immunodeficient mice were adoptively transferred highly purified human IgM ⁺ mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM ⁺ mBc are phenotypically diverse and significantly enriched in the IgM ^hi IgD ^low subset. Thus, RV-IgM ⁺ mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo , and can mediate systemic antiviral immunity.