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Repeated cycles of dextran sulfate sodium (DSS) administration in mice, inducing chronic relapsing colitis, have been used to mimic human ulcerative colitis (UC). However, no systematic characterization of pro-inflammatory cytokines in these DSS mice has been reported. In this study, the development of colitis was examined by assessment of the disease severity and inflammation in the colon of C57BL/6 mice that received DSS. ELISA was used to analyze the levels of pro-inflammatory cytokines in serum, colon, spleen and supernatant of cultured splenocytes. mRNA levels of the above cytokines in colon and mesenteric lymph node (MLN) were measured with RT-PCR. The mice receiving three cycles of 2% DSS over a 43-day period showed a fluctuating appearance of diarrhea and bloody feces, and a significant reduction in body weight and colon length. When compared with normal control mice, an increase in TNF-α level in serum was detected in the DSS mice, along with a decrease in the amounts of TNF-α, IL-17, IL-1β and IL-6 in the colonic tissue. However, mRNA levels of these cytokines were found to be significantly increased in the colon while decreased in the MLN of the colitis mice. Further, the ELISA assay suggested a pronounced increase of TNF-α production by cultured splenocytes with PMA/ionomycin re-stimulation but no increase in its presence in spleen tissue upon DSS challenge. In conclusion, we have systematically demonstrated the dysregulation of pro-inflammatory cytokines in the DSS-induced chronic relapsing colitis model, which will provide markers to test emerging therapeutic strategies by this model.The mice receiving three cycles of 2% dextran sodium sulfate (DSS) showed an increase in amount of TNF-α in serum.Amounts of TNF-α, IL-17 and IL-1β in colonic tissue of the colitis mice were decreased, while their mRNA levels were significantly increased when compared with those of normal control mice.A pronounced increase of TNF-α production in cultured splenocytes but no increase in spleen tissue was detected upon DSS challenge.We demonstrated the dysregulation of pro-inflammatory cytokines in the DSS-induced mice chronic relapsing colitis model.