ETHNOPHARMACOLOGICAL RELEVANCE: Zataria multiflora Boiss. (Shirazi thyme) is traditionally used for digestive disorders and inflammatory conditions. Despite its known anti-inflammatory, immunomodulatory, and antioxidant properties, there is limited clinical evidence on its efficacy for ulcerative colitis (UC). AIM OF THE STUDY: To evaluate the effectiveness of Zataria multiflora Boiss. (Z. multiflora) extract in alleviating UC symptoms, reducing inflammatory markers, and modulating immune-related gene expression. MATERIALS AND METHODS: In a multicenter, randomized, placebo-controlled, triple-blind trial in Iran, 92 participants received Z. multiflora extract (6 mg/kg/day) or a placebo for two months. Inflammatory markers and gene expression were analyzed from blood samples. Disease activity was assessed using the Partial Mayo Score (p-Mayo) and the Gastrointestinal Symptom Rating Scale (GSRS). Data were analyzed with SPSS software. RESULTS: The Z. multiflora group showed significant reductions in C-reactive protein (CRP) (p <
0.001), Interleukin-17 (IL-17) (p = 0.001), Interferon-gamma (IFN-γ) (p = 0.002), Nuclear Factor kappa B (NF-κB) (p = 0.002), T-box Transcription Factor T-bet (T-bet) (p = 0.006), and Retinoic Acid-Related Orphan Receptor gamma t (ROR-γt) (p <
0.001). No significant changes were observed in Erythrocyte Sedimentation Rate (ESR) (p = 0.25), GATA Binding Protein 3 (GATA3) (p = 0.09), and Forkhead Box P3 (FOXP3) (p = 0.17). Symptoms such as heartburn, acid reflux, bloating, diarrhea, and fecal urgency improved (p <
0.05). The GSRS score improved (p <
0.001), while the p-Mayo score did not show a significant change (p = 0.24). CONCLUSION: Z. multiflora extract significantly alleviated UC symptoms and reduced inflammatory markers, indicating its potential as a complementary treatment for UC. However, the study was limited by its short intervention period and the absence of biopsy analysis to assess local tissue effects. Further longitudinal studies are required to validate these findings and determine long-term efficacy.