Dysbiosis causes continuous progress of inflammatory bowel disease (IBD). Herein, we aim to explore whether Salidroside (Sal), which is a major glycoside extracted from Rhodiola rosea L., could ameliorate dextran sulfate sodium (DSS)-induced colitis by modulating the microbiota. Results showed that oral treatment with 15 mg kg-1 of Sal inhibited DSS-induced colitis in mice as evidenced by colon length, histological analysis, disease activity index (DAI) score, and the proportion and number of macrophages in the intestine. The gut microbiota of colitic mice was also partly restored by Sal. A fecal microbiota transplantation (FMT) study was designed to verify the causality. Compared with DSS-treated mice, FM from the Sal-treated donor mice significantly mitigated the symptoms of colitic mice, including reducing the DAI score, alleviating tissue damage, boosting the expression of mucin protein (mucin-2) and tight junction (TJ) proteins (occludin and zonula occludens-1 (ZO-1), and decreasing M1 macrophages in the gut. It was found that both Sal and FMT affected the structure and abundance of the gut microbiota as reflected by the decreased relative abundance of Turicibacter, Alistipes, Romboutsia and the increased relative abundance of Lactobacillus at the genus level. Moreover, the anti-inflammatory effect of Sal disappeared when the gut microbiota was depleted by antibiotics, demonstrating that Sal alleviated the intestinal inflammation in a gut microbiota-dependent manner. Thus, Sal could be a remarkable candidate as a functional food for colitis.