The present study was performed to evaluate the effect of intestinal microbiota transplantation (IMT) on intestinal flora and inflammatory factor levels in patients with ulcerative colitis (UC). In this study, 94 UC patients who attended the Department of Proctology or the Department of Gastroenterology departments of Sinopharm Dongfeng General Hospital between April 2021 and April 2022 were identified as research participants and were assigned to the control or Research Groups via the random number table method, with 47 cases in each group. Interventions included oral mesalamine for patients in the control group and oral mesalamine plus IMT for those in the research group. Outcome measures included clinical efficacy, intestinal microbiota score, enteroscopy score, Sutherland index, inflammatory factor level, intestinal mucosal barrier function level, and adverse reactions. Mesalamine plus IMT was associated with significantly higher treatment efficiency (97.8%) versus mesalamine alone (80.85%) (P<0.05). Mesalamine plus IMT provided a better intestinal microbiota balance and milder disease symptoms versus mesalamine, as evidenced by the significantly lower intestinal microbiota scores, colonoscopy scores, and Sutherland index (P<0.05). In post-treatment, patients with IMT exhibited more mitigated inflammatory responses than those without, as shown by the higher levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-17 (IL-17), and interleukin-23 (IL-23) (P<0.05). Significantly lower D-lactate and serum diamine oxidase (DAO) levels were observed after IMT intervention than those with mesalamine alone (P<0.05). IMT features no significant increase in adverse effects than the control group (P>0.05). IMT efficiently ameliorates the intestinal microbiota conditions of UC patients, mitigates inflammatory responses in the body, and facilitates the restoration of intestinal mucosal barrier function with no significant increase in adverse effects.