Rotavirus infection of children genetically predisposed to type 1 diabetes may accelerate disease progression. Supporting this, oral infection of adult NOD mice with rhesus monkey rotavirus (RRV) accelerates diabetes onset, without pancreatic infection. Diabetes onset is more rapid in the RRV-infected mice showing the greatest T helper 1 bias in rotavirus-specific IgG. RRV infection increases TNF mRNA expression in islets and pancreatic lymph nodes (PLN), and IFNγ mRNA expression in PLN. Infectious RRV is found in PLN and the non-T, non-B cell compartment of mesenteric lymph nodes (MLN). Intracellular RRV is associated with a population of MHC II+ antigen-presenting cells, including CD11c+ dendritic cells. Antigen-presenting cells containing RRV express increased MHC I and II levels. The aim of this study was to further elucidate the mechanisms involved in RRV acceleration of diabetes onset in NOD mice. We showed that B cells in MLN and PLN upregulated MHC I expression, and T cell expression of IFNγ and TNF was increased. Infection with CRW-8 rotavirus, which does not accelerate diabetes in NOD mice, did not activate B cells. Furthermore, pro-inflammatory cytokine expression was unaltered in RRV-infected young adult NOD mice, which do not show accelerated diabetes onset. Thus, cytokine expression and B cell activation appeared to be associated with diabetes acceleration. B cells from RRV-infected mice showed an enhanced ability to induce auto-reactive T cell proliferation ex vivo. RRV exposure ex vivo induced the activation of purified B cells and T cells activation in the presence of sorted plasmacytoid dendritic cells via signalling through the type 1 IFN receptor. Thus, RRV infection may lead to type 1 IFN-specific lymphocyte activation, and autoreactive T cell activation by B cells, in the MLN and PLN. Overall, these data provide evidence for bystander activation as one mechanism leading to murine diabetes acceleration by rotavirus.