Background and Objectives: The exosomes released by mesenchymal stromal cells (MSCs) in classical FBS-containing media have been demonstrated as an alternative, cell-free therapy in various diseases including inflammatory bowel disease (IBD). It has been found that the function of exosomes is affected by culture condition. We previously developed a serum-free, xeno-free and chemically defined medium, and umbilical cord-derived MSCs in this medium retained the immunosuppressive capability. Methods: In this study, we evaluated the immunosuppressive function of exosomes from MSCs (MSC-Exo) in defined medium and their therapeutic effect on treating colitis. Results and Conclusions: In vitro studies indicated that MSC-Exo reduced the concentration of pro-inflammatory cytokines UN- 7, TNF- alpha and IL-1 beta, and increased the secretion of anti-inflammatory cytokines TGF- beta 1 and IL-10, but no significant change of inhibitory effect on peripheral blood mononuclear cells proliferation was shown. In vivo experimental colitis showed that administration of MSC-Exo was able to significantly ameliorate the disease activity index score, weight loss, colon shortening, and the histological colitis score through up-regulation anti-inflammatory responses and down-regulation of inflammatory responses. Moreover, the use of MSC-Exo (200 mu g) led to an improved therapeutic efficacy when compared with MSCs at a dose of 1 x 10(6) cells. Our findings indicate that the exosomes from MSCs in defined medium possess a certain degree of immunosuppressive effect in vitro and exhibit a therapeutic capability in a mouse model of DSS-induced colitis through suppressing inflammation mechanism.