Rotavirus (RV) causes significant morbidity and mortality in children worldwide. The intestinal microbiota plays an important role in modulating host-pathogen interactions, but little is known about the impact of commonly used probiotics on human RV (HRV) infection. In this study, we compared the immunomodulatory effects of Gram-positive [Lactobacillus rhamnosus strain GG (LGG)] and Gram-negative [Escherichia coli Nissle (EcN)] probiotic bacteria on virulent human rotavirus (VirHRV) infection and immunity using neonatal gnotobiotic (Gn) piglets. Gn piglets were colonized with EcN, LGG, EcN+LGG or uncolonized and challenged with VirHRV. Mean peak virus shedding titers and mean cumulative fecal scores were significantly lower in EcN-colonized compared to LGG-colonized or uncolonized piglets. Reduced viral shedding titers were correlated with significantly reduced small intestinal HRV IgA antibody responses in EcN-colonized compared to uncolonized piglets post-VirHRV challenge. However the total IgA levels post-VirHRV challenge in the intestine and pre-VirHRV challenge in serum were significantly higher in EcN-colonized than in LGG-colonized piglets. In vitro treatment of mononuclear cells (MNCs) with these probiotics demonstrated that EcN, but not LGG, induced IL-6, IL-10, and IgA, with the latter partially dependent on IL-10. However, addition of exogenous recombinant porcine IL-10 + IL-6 to MNCs co-cultured with LGG significantly enhanced IgA responses. The greater effectiveness of EcN in moderating HRV infection, may also be explained by the binding of EcN, but not LGG to Wa HRV particles or HRV 2/4/6 virus-like particles (VLP) but not 2/6 VLP. Results suggest that EcN and LGG differentially modulate RV infection and B cell responses.