Plasmacytoid dendritic cells (pDCs) are highly specialized APCs that, in addition to their well-recognized role in anti-viral immunity, also regulate immune responses. Liver-resident pDCs are considerably less immunostimulatory than those from secondary lymphoid tissues and are equipped to promote immune tolerance/regulation through various mechanisms. IL-27 is an IL-12 family cytokine that regulates the function of both APCs and T cells, although little is known about its role in pDC immunobiology. In this study, we show that mouse liver pDCs express higher levels of IL-27p28 and EBV-induced protein 3 (Ebi3) compared with those of splenic pDCs. Both populations of pDCs express the IL-27Rα/WSX-1; however, only liver pDCs significantly upregulate expression of the coregulatory molecule B7 homolog-1 (B7-H1) in response to IL-27. Inhibition of STAT3 activation completely abrogates IL-27-induced upregulation of B7-H1 expression on liver pDCs. Liver pDCs treated with IL-27 increase the percentage of CD4(+)Foxp3(+) T cells in MLR, which is dependent upon expression of B7-H1. pDCs from Ebi3-deficient mice lacking functional IL-27 show increased capacity to stimulate allogeneic T cell proliferation and IFN-γ production in MLR. Liver but not spleen pDCs suppress delayed-type hypersensitivity responses to OVA, an effect that is lost with Ebi3(-/-) and B7-H1(-/-) liver pDCs compared with wild-type liver pDCs. These data suggest that IL-27 signaling in pDCs promotes their immunoregulatory function and that IL-27 produced by pDCs contributes to their capacity to regulate immune responses in vitro and in vivo.