Background and purpose: In multiple sclerosis (MS) and its murine model, experimental autoimmune encephalomyelitis (EAE), chronic inflammation damages the myelin of central nervous system. Recently, interleukin-27 (IL-27) has been recognized as a feasible choice for treatment of autoimmune diseases such as MS due to its anti-inflammatory properties. However, the underlying mechanisms have not yet been specified clearly. The present study, investigated the immunomdulatory effects of IL-27 in C57BL/6 mice with EAE. Materials and methods: In this experimental study, two groups of EAE mice (test and control groups) received intraperitoneal injection of P240-mIL-27 (200 μ g) and P240 plasmid (200 μ g), respectively. The disease severity was evaluated daily for 30 days. At the end of the treatment period, the mice were sacrificed and the levels of IL-17, IFN-γ , IL-6, and IL-10 were measured in splenocytes culture media using ELISA method. Also, the percentage of CD4+Foxp3+ regulatory T cells (Treg) in spleen cells was analyzed using flow cytometry. Results: Severity of EAE significantly decreased in test group (P240-mIL27), compared to that of the control group. In test group, the levels of IL-17, IFN-γ , and IL-6 were significantly lower (P<0. 001), while IL-10 levels were significantly higher compared to those of the control group (P<0. 001). Moreover, the percentage of Treg cells in test group was significantly higher than that of the control mice (P<0. 001). Conclusion: IL-27 can be a suitable choice in treatment of inflammatory diseases such as MS via increasing Treg cells and IL-10, and suppression of inflammatory cytokines.