Intestinal and systemic dendritic cell (DC) frequencies, serum and small intestinal content cytokines and uptake/binding of human rotavirus (HRV) virus-like particles (VLP) were studied in HRV acutely infected or mock-inoculated neonatal gnotobiotic piglets. Intestinal, mesenteric lymph node (MLN) and splenic plasmacytoid DCs (pDCs), conventional DCs (cDCs) and macrophages/monocytes were assessed by flow cytometry. In infected pigs, serum and small intestinal content interferon-alpha (IFN-alpha) were highest, interleukin-12 (IL-12) was lower and IL-10, tumour necrosis factor-alpha and IL-6 were minimal. Compared with mock-inoculated piglets, frequencies of total intestinal DCs were higher; splenic and MLN DC frequencies were lower. Most intestinal pDCs, but few cDCs, were IFN-alpha(+) and intestinal macrophages/monocytes were negative for IFN-alpha. Serum IFN-alpha levels and IFN-alpha(+) intestinal pDCs were highly correlated, suggesting IFN-alpha production in vivo by intestinal pDCs (r=0.8; P<0.01). The intestinal pDCs and cDCs, but not intestinal macrophages/monocytes, of HRV-infected piglets showed significantly lower VLP uptake/binding compared with mock-inoculated piglets, suggesting higher activation of pDCs and cDCs in infected piglets. Both intestinal pDCs and cDCs were activated (IFN-alpha(+) and lower VLP binding) after HRV infection, suggesting their role in induction of HRV-specific immunity. Dose-effects of HRV on serum IFN-alpha and IFN-alpha(+) DCs were studied by infecting piglets with 100-fold higher HRV dose. A high dose increased parameters associated with inflammation (diarrhoea, intestinal pathology) but serum IFN-alpha and IFN-alpha(+) DCs were similar between both groups. The pDCs have both anti- and pro-inflammatory functions. Stimulation of the anti-inflammatory effects of pDCs after the high dose, without increasing their pro-inflammatory impacts, may be critical to reduce further immunopathology during HRV infection.