Background. Mesenchymal stem cells (MSC) based on their immune-modulatory and anti-inflammatory properties have been candidate as therapeutic treatment for several immune disease including inflammatory bowel disease (IBD). Although most of beneficial effects are explained by MSC engrafting at the site of tissue injury with modulation of inflammatory reactions, the mechanisms by which MSC exert their activities needed to be elucidated. Aim of this study was to investigate the mechanisms underlying the therapeutic efficacy of MSC in Dextran Sodium Sulphate (DSS) induced colitis. Methods. Colitis was induced in C57BL6 mice by 3% DSS treatment for 10 days. MSC were isolated from bone marrow of wild type (WT) and GFP-transgenic C57BL6 mice, cultured for 4 weeks and then sorted for Sca-1+, CD31-, cocktail lineage- surface markers. At day 5 of colitic induction the mice were treated intraperitoneally with a single injection of 3x106 GFP-MSC or with 5 daily injections of recombinant murine TNF?-stimulating gene protein 6 (TSG-6) (4 ?g). Body weight and disease activity index (DAI) were monitored daily and the damage of murine colonic mucosa was evaluated by endoscopic and histological scores. To follow the migratory activity of the MSC, GFP-MSC were injected intraperitoneally in healthy or colitic mice at day 5 and their presence was assessed by flow cytometry after 24 and 48 hours in the colon and mesenteric lymph nodes. Levels of TSG-6, IL-6 and IFN-? were measured in serum and in mucosal extracts by ELISA, while MMP activities were quantified by WB. Results. We found that one injected into the peritoneum MSC remain into the peritoneal cavity, where they aggregate along with macrophages and lymphocytes, generating organized structures. Only a small fraction (< 1%) of cells reached the inflamed colon. In vitro and in vivo assays have demonstrated that MSC secrete a multipotent anti-inflammatory protein TSG-6 able to reduce the damage to the colon. In fact, recombinant murine TSG-6 treatment improved survival rate and DSS-induced colitis by reducing markedly both systemic and mucosal levels of IL-6, IFN-?, neutrophil infiltration, and MMP activities. Conclusions. Overall, these data indicate that the MSC gut-homing is not relevant for exerting their immunomodulatory effects, but MSC dampen the mucosal inflammatory response at distance by releasing a potent anti-inflammatory protein.