Development of a safe, cheap and efficient vaccine against rotavirus is important to reduce the morbidity and mortality associated with gastroenteritis in infants worldwide. High quantities of two inner core rotavirus-derived proteins (VP2 and a nonglycosylated mutant VP6 (VP6(NG)) from the RF81 bovine strain) were produced in the milk of transgenic rabbits. We show here that rectal administration of partially purified milk-derived VP2 and VP6(NG) proteins with the detoxified LT(R192G) adjuvant almost completely prevented fecal shedding induced by a highly infectious challenge in mice with the murine ECw strain. The vaccine generated rotavirus-specific fecal secretory IgA, systemic IgG and IgA and a rotavirus-specific Th1 response. We thus demonstrate in clinically feasible settings that mass production of viral protein in transgenic milk is a promising way to generate subunit vaccine against rotavirus.