BACKGROUND: Plantamajoside (PMS) is a natural bioactive compound derived from medicinal, food homologous plants of the genus Plantago. PURPOSE AND METHODS: This study aimed to investigate the protective effects of PMS on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice and explore the associated mechanisms. RESULTS: We found that PMS treatment significantly alleviated UC symptoms in mice by preventing body weight loss, increasing colon length, and reducing disease activity index scores. Moreover, PMS alleviated colonic lesions, increased the number of goblet cells, upregulated the expression of intestinal barrier proteins (ZO-1, occludin, and claudin-3), and decreased the levels of pro-inflammatory factors. PMS treatment modulated the gut microbiota by increasing the relative abundance of Bacteroidota and Verrucomicrobiota and decreasing that of Firmicutes and Proteobacteria at the phylum level. At the genus level, PMS suppressed the abundance of pathogenic bacteria, such as Turicibacter and upregulated the abundance of [Eubacterium]_xylanophilum_group. Fecal microbiota transplantation experiments further confirmed that PMS treatment alleviated UC by modulating the gut microbiota. Transcriptomic analysis of colon tissues, coupled with reverse transcription-quantitative polymerase chain reaction and western blotting, showed that PMS treatment upregulated cystathionine beta-synthase (CBS) expression and inhibited NF-kappaB pathway activation. In a lipopolysaccharide-induced inflammation model in RAW264.7 cells, PMS treatment inhibited the secretion of pro-inflammatory cytokines, upregulated CBS expression, and prevented NF-kappaB pathway activation. CONCLUSION: PMS protects against UC in mice via multiple mechanisms, including modulating the gut microbiota, increasing the expression levels of CBS, and inhibiting the NF-kappaB pathway.