Ulcerative colitis (UC) prevalence is rising globally, yet fewer than 50% of patients achieve mucosal healing (MH) with first-line 5-aminosalicylic acid (5-ASA) therapy. We aimed to identify microbial signatures that could predict the treatment efficacy of 5-ASA. Active UC patients on standardized 5-ASA treatment were prospectively enrolled. Shotgun metagenomic sequencing was performed to identify the taxonomic and functional profiles before and after treatment. Six species were enriched in the effective group and 3 species in the ineffective group at baseline. Faecalibacterium prausnitzii, Blautia massiliensis, and Phascolarctobacterium faecium were consistently depleted in the ineffective group at both time points. A random forest model based on these three species predicted ineffective 5-ASA treatment with area under the curve (AUC) = 0.80 (validation in the Inflammatory Bowel Disease Multi'omics Database [IBDMDB]: AUC = 0.82, specificity = 0.88, negative predictive value [NPV] = 0.70, and positive predictive value [PPV] = 0.80). Gut microbiome signatures have potential to serve as non-invasive predictors for ineffective 5-ASA treatment in UC.