Mesenchymal stem cells (MSCs) reportedly inhibit the mixed lymphocyte reaction. Whether this effect is mediated by dendritic cells (DCs) is still unknown. In this study, we used an in vitro model to observe the effects of MSCs and their supernatants on the development of monocyte-derived DCs. Phenotypes and the endocytosic ability of harvested DCs were determined by flow cytometry; interleukin 12 (IL-12) secreted by DCs was evaluated by enzyme-linked immunosorbent assay (ELISA); and the antigen-presenting function of DCs was evaluated by MLR. Our results show that MSCs inhibit the up-regulation of CD1a, CD40, CD80, CD86, and HLA-DR during DC differentiation and prevent an increase of CD40, CD86, and CD83 expression during DC maturation. MSCs supernatants had no effect on DCs differentiation, but they inhibited the up-regulation of CD83 during maturation. Both MSCs and their supernatants interfered with endocytosis of DCs, decreased their capacity to secret IL-12 and activate alloreactive T cells. Thus, effects of MSCs on DCs contribute to immunoregulation and development.