Gastrodin (GAS), the principal bioactive composition of Gastrodia elata Blume, has potential for pharmaceutical applications. Several studies in recent years have shown that GAS may enhance neurotrophic benefits, reduce inflammation, and act as an antioxidant. In this study, we sought to identify the molecular mechanisms underlying the protective benefits of GAS against colitis induced by dextran sodium sulfate (DSS) in mice. GAS (200 mg/kg) significantly ameliorated the severity of colitis in mice caused by DSS, as evidenced by an increase in colon length, a reduction in disease activity index, a decrease in tissue damage, and a reduction in body weight loss. Additionally, GAS inhibited DSS-induced hyperactivation of inflammation-related NF-κB signaling pathways to reduce the production of inflammatory mediators, thereby mitigating the inflammatory response in mice. Furthermore, the administration of GAS restored the function of the gastrointestinal barrier by increasing the count of goblet cells, as well as the levels of tight junctionassociated proteins, including Zonula occludens-1 (ZO-1), Occludin, and Claudin-3. GAS also influenced the overall richness of the gut microbiota, as shown by 16S rRNA sequencing analysis, consequently boosting the proliferative rate of probiotic species, such as Lachnospiraceae and Muribaculaceae, while reducing the richness of harmful bacteria including Escherichia_Shigella, Enterobacteriaceae, Bacteroidaceae, and Bacteroides. GAS (200 mg/kg) alleviated ulcerative colitis (UC) by modulating gut dysbiosis, as demonstrated by a fecal microbial transplantation (FMT) test. Furthermore, inflammatory damage induced by lipopolysaccharide (LPS) was averted in RAW264.7 cells by GAS administration, hence preventing the NF-κB signaling pathway from being activated in these experimental conditions conducted in vitro. Overall, the data indicate that GAS treatment effectively reduces colitis caused by DSS by regulating gut microbiota, suppressing inflammation, and preserving the mucosal barrier integrity.