Indigo naturalis (also known as Qing-dai, or QD), a traditional Chinese medicine, has been widely used as an anticolitis regimen in the clinical practice of Chinese medicine. However, the precise mechanisms behind its efficacy remain unknown. We investigated the protective effects and associated molecular mechanisms of QD in DSS-induced colitis in mice. We found that QD administration attenuated DSS-induced colon shortening, tissue damage, and the disease activity index during the onset of colitis. Moreover, QD administration significantly suppressed colonic MPO activity and increased the activities of colonic T-SOD, CAT, and GSH-Px, as well the expression of p-AMPK and Nrf-2 in colon tissues of colitic mice. In addition, QD was capable of reducing the colonic Th1 and Th17 cell cytokines, the frequencies of Th1 and Th17 cells, and the phosphorylation of p-STAT1 and p-STAT3 in the mesenteric lymph nodes of colitic mice. An in vitro assay showed that QD significantly suppressed the differentiation of Th1 and Th17 cells. These findings suggest that QD has the potential to alleviate experimental colitis by suppressing colonic oxidative stress and restraining colonic Th1/Th17 responses, which are associated with activating AMPK/Nrf-2 signals and inhibiting STAT1/STAT3 signals, respectively. These findings also support QD as an effective regimen in the treatment of IBD. Copyright © 2019 Hai-tao Xiao et al.