Tonsil-derived mesenchymal stem cells (TMSC) have characteristics of MSC and have many advantages. In our previous studies, intraperitoneal (IP) injection of TMSC in acute and chronic colitis mouse models improved the disease activity index, colon length, and the expression levels of proinflammatory cytokines. However, TMSC were not observed to migrate to the inflammation site in the intestine. The aim of this study was to verify the therapeutic effect of conditioned medium (CM) released by TMSC (TMSC-CM) in a mouse model of dextran sulfate sodium (DSS)-induced chronic colitis. TMSC-CM was used after seeding 5×105 cells onto a 100 mm dish and culturing for 5–7 days. TMSC-CM was concentrated (TMSC-CM-conc) by three times using a 100 kDa cut-off centrifugal filter. Seven-week-old C57BL/6 mice were randomly assigned to the following 5 groups: 1) normal, 2) colitis, 3) TMSC, 4) TMSC-CM, and 5) TMSC-CM-conc. Chronic colitis was induced by continuous oral administration of 1.5% dextran sulfate sodium (DSS) for 5 days, followed by 5 additional days of tap water feeding. This cycle was repeated two more times (total 30 days). Phosphate buffered saline (in the colitis group), TMSC, TMSC-CM, and TMSC-CM-conc were injected via IP route 4, 4, 12, and 4 times, respectively. Reduction of disease activity index, weight gain, recovery of colon length, and decreased in the expression level of the proinflammatory cytokines, interleukin (IL)-1β, IL-6, and IL-17 were observed at day 30 in the treatment groups, compared to control. However, histological colitis scoring and the expression level of tumor necrosis factor α and IL-10 did not differ significantly between each group. TMSC-CM showed an equivalent effect to TMSC related to the improvement of inflammation in the chronic colitis mouse model. The data obtained support the use of TMSC-CM to treat inflammatory bowel disease without any cell transplantation.