CD83 is up-regulated on the surface of dendritic cells (DCs) during maturation and has been widely used as a marker for mature DCs. Recently, we reported the recombinant expression of the extracellular immunoglobulin domain of human CD83 (hCD83ext). Using this soluble form of CD83, allogeneic as well as specific cytotoxic T lymphocyte proliferation could be blocked in vitro. Here we report the functional analysis of soluble CD83 in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as a model. Strikingly, only three injections of soluble CD83 prevented the paralysis associated with EAE almost completely. In addition, even when the EAE was induced a second time, CD83-treated mice were protected, indicating a long-lasting suppressive effect. Furthermore, soluble CD83 strongly reduced the paralysis in different therapeutic settings. Most important, even when the treatment was delayed until the disease symptoms were fully established, soluble CD83 clearly reduced the paralyses. In addition, also when EAE was induced a second time, soluble CD83-treated animals showed reduced disease symptoms. Finally, hCD83ext treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In summary, this work strongly supports an immunosuppressive role of soluble CD83, thereby indicating its therapeutic potential in the regulation of immune disorders in vivo.