Bifidobacterium species (B. bifidum and B. infantis), with or without prebiotic compounds (arabino-galactan, short-chain fructo-oligosaccharide, iso-malto-dextrins), were orally fed to Balb/c pups (n = 192) to evaluate their potential synergistic effects on modulating the course of rhesus rotavirus (RRV) infection, as well as their ability to mediate the associated mucosal and humoral immune responses. Rotavirus-specific IgA and IgG in serum, rotavirus antigen, and specific IgA in feces were measured by ELISA. Mucosal total IgA and IgG levels were determined in Peyer's patches by flow cytometry. Significantly delayed onset (p = 0.001) and early resolution (p < 0.001) of diarrhea were observed in bifidobacteria-treated, RRV-infected mice compared with RRV-infected control mice. Supplementation with prebiotic compounds did not shorten the clinical diarrhea course more than that observed with bifidobacteria treatment alone. Rotavirus-specific IgA in feces was 16-fold elevated on d 5 postinfection in bifidobacteria-treated, RRV-infected mice compared with the RRV-infected alone group. In addition, the level of rotavirus-specific IgA in serum was four-fold higher in bifidobacteria-treated, RRV-infected litters versus mice challenged with RRV alone on 28 and 42 d postinfection. No enhancement of the immune response was found in RRV-infected mice that were treated with both bifidobacteria and prebiotic compounds over those treated with bifidobacteria only. The findings suggest that bifidobacteria may act as an adjuvant by modulating early mucosal and strong humoral rotavirus-specific immune responses, and mitigate severity of rotavirus-induced diarrhea.