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Abstract Kale has been reported to have potential alleviating effects on colitis. However, the functional activities of kale dietary fibers and kale flavonoids (KF), as well as the effects of their combined use, were currently unknown. In this study, the efficacy of whole kale, kale soluble dietary fiber (KSDF), kale insoluble dietary fiber (KIDF), KF, mixture of KSDF and KF, and mixture of KIDF and KF on dextran sulfate sodium‐induced colitis mice was assessed to dissect which component(s) play the key role in alleviating colitis. The study demonstrated that administration of KSDF, KIDF, or KF resulted in various degrees of beneficial effects, including reduction of weight loss, colonic shortening, disease activity index score, and mRNA levels of inflammatory factors (TNF‐α, IL‐6, IL‐1β, and IL‐33), increase of mucin expression and suppression of intestinal g_Sutterella, g_Parabacteroides, f_Enterobacteriaceae relative abundance. However, 20% mouse mortality was found in group of KSDF combined with KF. Further analysis found that the combination antagonized the alleviating effects of both agents alone on colitis in mice. It is the first to report that there is antagonism between KSDF and KF in colitis, and the regulation of intestinal flora may be the mechanism. In addition, this study also showed that KSDF, KIDF, and KF alone had excellent effects to alleviate colitis. This study provides a new perspective and evidence to explore the effect of interaction among different food nutrients on intestinal inflammation.