To explore the ameliorative effect and mechanism of gastrodigenin (4-hydroxybenzyl alcohol, HBA) on ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) in mice, 50 male C57BL/6J mice were randomly divided into a blank control group, a model group, and three HBA intervention groups (15, 30, 60 mg/kg). Except for the blank control group, the other groups were given DSS in drinking water to establish the UC model for seven consecutive days. Meanwhile, HBA intervention was conducted. Specifically, the blank control and model groups were intragastrically administered 1% carboxymethyl cellulose solution, while the HBA intervention groups were intragastrically administered HBA solution at corresponding doses for seven days. After the intervention, the body weight, colon length of the mice were measured, and the disease activity index (DAI) was evaluated. HE staining was used to observe the pathological morphology. The contents of inflammatory factors interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in the serum were detected by ELISA. The mRNA expression levels of inflammatory factors IL-6, IL-1β, TNF-α, and tight junction proteins zonula occluden-1 (ZO-1), Claudin-1, and Occludin in the colon tissue were detected by real-time fluorescence quantitative PCR. The levels of oxidative stress markers superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were detected by kits. The expression of tight junction proteins ZO-1, Claudin-1, and Occludin in the colon of mice was detected by Western blot. 16S rRNA sequencing was used to analyze the composition and differences of intestinal flora. The results showed that HBA intervention significantly improved the severity of UC induced by DSS, such as weight loss, colon atrophy, increased DAI score, and tissue damage. In addition, HBA alleviated the inflammatory response in UC mice, relieved oxidative stress, and restored the function of the intestinal barrier by increasing the levels of tight junction proteins, with the high-dose group being the most significant. Therefore, the high-dose HBA group was selected for 16S rRNA sequencing. The analysis showed that the composition and abundance of intestinal flora in UC mice were significantly different from those in the blank control group. HBA could improve the structure of intestinal flora and inhibit the proliferation of pathogenic bacteria Turicibacter and Bacteroides. It is indicated that HBA improves the symptoms of DSS-induced UC by inhibiting inflammation, relieving oxidative stress, maintaining the integrity of the mucosal barrier, and regulating the intestinal microbiota.