Using a rat dextran sulfate sodium (DSS)-colitis model, we elucidated that the expression of miRNAs in colorectal tissues, plasma, and feces, particularly miR-31a-5p, miR-181b-5p, and miR-223-3p, could be used as noninvasive biomarkers to evaluate the reversibility of the model. We further investigated whether changes in miRNA levels were reproducible in chronic DSS-induced colitis in rats. Male SD rats were administered 5% DSS in drinking water for two cycles. Cycle 1 consisted of a 7-d dosing period and 14-d recovery period, followed by Cycle 2 consisting of a 5-d dosing period and 7-d recovery period. In-life parameters and the disease activity index (DAI) were respectively examined or calculated daily. Colon length and pathological changes were assessed postmortem in Cycle 2. A panel of nine miRNAs was also measured in colorectal tissues, plasma, and feces using digital PCR. The changes in DAI score and colon length were evident in Cycle 2. Erosive and inflammatory changes were observed in the colon and rectum following DSS treatment. At the end of the off-dose period of Cycle 2, the histological changes in the rectum worsened, while the colon changes showed recovery. The expression patterns of all miRNAs were almost the same in Cycle 2 when compared to those in a previous study (Kodama et al., 2021). Fecal miR-223-3p could be also a useful non-invasive indicator to evaluate the reversibility in chronic DSS-induced colitis in rats.