Glucocorticoids (GCs) are widely used as anti-inflammatory and immunosuppressive agents. Effects of GC have mainly been attributed to the suppression of T cells. Recently, several studies have indicated the role of dendritic cells (DC) in GC-mediated immunosuppression. We investigated the effect of GC on characteristics of DC. Given the crucial role of Toll-like receptor (TLR) triggering for the initiation of DC maturation program, we analyzed the expression of TLR2, 3, 4 by GC-treated DC. To extend our in vitro findings, we analyzed the distribution of DC subsets in the blood of patients treated with high-dose corticosteroids. DC differentiation in presence of GC was skewed to a qualitatively distinct population incapable of inducing an efficient immune response, whereas GC presence during the process of maturation significantly reduced DC IL-12 p70 and TNF production and T cell stimulatory function. Despite the fact that GC increased expression of TLR2, 3 and 4 on DC, their stimulation with TLR-derived signals did not induce maturation. Administration of high-dose GC to the patients with systemic autoimmunity induced a decrease of circulating myeloid DC and abrogated plasmacytoid DC. These findings provide further insights into the mechanisms of GC immunosuppressive functions and reveal additional mechanisms of their therapeutic efficiency.