Microbially modified bile acids shape host physiology by regulating nutrient absorption, glucose homeostasis, circadian rhythms and thermoregulation. Here we identify a previously unrecognized drug-activating function of microbial bile acid conjugation. By systematically mining human LC–MS/MS datasets across public repositories and linking uncharacterized bile acid spectra to health-associated metadata, we discovered conjugates of the >75-year-old anti-inflammatory drug 5-aminosalicylic acid (5-ASA) with primary and secondary bile acids, including cholic, deoxycholic and lithocholic acids. These bile acid–drug conjugates were detected specifically in individuals treated with 5-ASA or its prodrugs. Multiple gut bacteria, including members of the Bacteroidota and Bacillota, generated cholyl–5-ASA in vitro, and bile salt hydrolase–associated transaminase activity was required for conjugate formation. In a mouse model of colitis, cholyl–5-ASA was associated with reduced intestinal inflammatory pathology and showed markedly enhanced activation of PPAR-γ in cell-based reporter assays compared with 5-ASA alone. Consistent with this activity, cholyl–5-ASA elicited selective immunophenotypic changes in CD4⁺ T cells in vitro, including increased Foxp3+ regulatory T cells. Together with prior evidence that 5-ASA efficacy depends on the microbiome, these findings support a model in which microbial bile acid conjugation represents a key activation step for 5-ASA therapy. More broadly, this work demonstrates how pan-repository metabolomics can uncover previously unrecognized microbiome-dependent chemical functions with direct therapeutic relevance.
