Under normal conditions, plasmacytoid dendritic cells (pDCs) are located in peripheral lymphoid organs or circulate in the blood, from where they can migrate to sites of infection or inflammation. In inflamed tissues, pDCs can be exposed to elevated levels of reactive oxygen species produced by inflammatory cells and we presume that oxidative stress could affect the cellular responses of pDCs to microenvironmental stimuli. To explore this possibility, human pDCs isolated from peripheral blood of healthy donors were treated with H₂O₂ and R837 (a Toll-like receptor 7 ligand), separately and in combination. Our results demonstrate that treatment with a low concentration (0.01μM) of H₂O₂ resulted in only slight changes in the expression of CD40, CD80, CD86, and CD83; however, low-dose H₂O₂ markedly decreased the expression of HLA-DQ on pDCs. Exposure to H₂O₂ did not trigger the release of IL-6, TNF-α, IL-8, or IFN-α from pDCs. Although addition of H₂O₂ did not modify the capacity of pDCs to activate allogeneic IL-17- or IFN-γ-producing T cells, it significantly increased the ability of pDCs to stimulate IL-4-secreting T cells. Exposure of pDCs to H₂O₂ before cocultivation with naïve autologous T cells significantly lowered IL-10 production by T cells, but did not affect IL-17 release. It was also observed that H₂O₂-exposed pDCs provided stronger stimuli for Th2 than for Th1 differentiation upon autologous activation, compared to untreated pDCs, possibly because of elevated surface expression of OX40-L. Most importantly, when pDCs were stimulated with R837 in the presence of H₂O₂, decreased phenotypic activation, decreased chemokine and cytokine release, and impaired allo- and autostimulatory functions of pDCs were detected, indicating that pDCs exposed to oxidative stress in vivo may have an anti-inflammatory or tolerogenic role in regulating adaptive immune responses.