Black rice, as a dietary supplement, has received increasing attention because of its beneficial health properties. Although black rice and its major ingredients have been considered a potential therapy for ulcerative colitis (UC), its effect and underlying mechanism of action remain obscure. In this study, black rice was demonstrated to improve dextran sulfate sodium salt (DSS)-induced UC and restore intestinal barrier dysfunction. Cyanidin 3-O-glucoside (C3G) is the most abundant ingredient in black rice. Similarly, in vivo and in vitro studies have demonstrated that C3G alleviates colitis and intestinal barrier dysfunction. Proteomic analysis showed that C3G significantly reduced abnormally elevated swiprosin-1 levels in mice with colitis. Furthermore, the effect of C3G on mitigating colitis was inhibited after swiprosin-1 was overexpressed in intestinal tissue in vivo and Caco-2 cells in vitro. Acyl-CoA synthetase long-chain family member 1 (ACSL1) was identified as a potential protein that interacts with swiprosin-1 by co-immunoprecipitation and liquid chromatography-tandem mass spectrometry. By integrating metabolomics experiments and bioinformatics analysis of single-cell sequencing, ACSL1 was found to selectively bind to swiprosin-1 and regulate downstream linoleic acid metabolism in colitis. Moreover, C3G was observed to prevent the localization of ACSL1 within mitochondria via swiprosin-1, thereby inhibiting the oxidation of linoleic acid. This study demonstrated that C3G targeted swiprosin-1 to ameliorate UC via ASCL1-mediated linoleic acid metabolism, thereby providing a novel potential insight into the mechanism of C3G protection against colitis and establishing the groundwork for its clinical application.