Toll-like receptors (TLR) play an important role in the recognition of microbes by host sentinel cells that leads to the subsequent innate and adaptive immune responses. In this study, we evaluated the patterns of TLR2-, TLR3- and TLR9-expressing antigen presenting cells (APCs) in spleen and blood of gnotobiotic (Gn) pigs after colonization with a mixture of two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and Lactobacillus reuteri or infection with the virulent human rotavirus (HRV) Wa strain. We also assessed the influence of LAB on TLR and serum innate cytokine responses induced by HRV. Distributions of subpopulations of APCs [CD14+/-SWC3+CD11R1- monocytes/macrophages and CD14+/-SWC3+CD11R1+ conventional dendritic cells (cDCs)] were described in our previous report (Zhang, W., Wen, K., Azevedo, M.S., Gonzalez, A.M., Saif, L.J., Li, G., Yousef, A.E., Yuan, L., 2008. Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs. Vet. Immunol. Immunopathol. 121, pp. 222-231). We demonstrated that LAB induced strong TLR2-expressing APC responses in blood and spleen, HRV induced a TLR3 response in spleen, and TLR9 responses were induced by either HRV (in spleen) or LAB (in blood). LAB and HRV have an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of LAB. Overall, the frequencies of TLR-expressing CD14+ APCs were higher than CD14- APCs. LAB enhanced the IFN-gamma and IL-4 responses in serum, but it had a suppressive effect on the TLR3- and TLR9-expressing CD14- APC responses in spleen and the serum IFN-alpha response induced by HRV. These results elucidated the systemic TLR2-, TLR3-, and TLR9-expressing monocyte/macrophage and cDC responses after HRV infection, LAB colonization, and the two combined. Our findings facilitate the understanding of the mechanism of LAB's adjuvant effect on rotavirus vaccines and the diverse innate and adaptive immune responses induced by commensal LAB colonization versus rotavirus infection and the interactions between them.