Abstract Humoral immunity to rotavirus, an intestinal pathogen, confers protection against re-infection. We utilize both the human in vitro and murine in vivo models of rotavirus infection to delineate the role of primary plasmacytoid dendritic cells (pDCs) in initiating B cell responses. Here, we demonstrate that primary human pDCs are necessary and sufficient for B cell activation by rotavirus. This requires very few pDCs, indicating the potency of the stimulus. Intact type I interferon (IFN) recognition by B cells is essential for activation. We extend these findings in vivo, demonstrating that murine pDCs and IFNα/β also mediate B cell activation following oral rotavirus infection. Furthermore, rotavirus-specific antibody responses are defective in mice lacking functional pDCs at the time of infection. These data demonstrate, for the first time, that B cell activation and virus-specific antibody secretion in response to mucosal viral infection occur as a direct result of pDC-derived type I IFN. Importantly, we observe increased viral shedding in pDC-deficient mice, indicating that pDC-instructed B cells impact viral clearance. We conclude that pDCs, through rotavirus recognition and subsequent type I IFN production, critically influence the course of rotavirus infection. Thus, mucosal pDCs can display powerful adjuvant properties to initiate humoral immunity. Our findings may extend to other mucosal pathogens, such as HIV or influenza.