Abstract: This study aimed to establish a systematic framework for enhancing the bioconversion efficiency of ellagic acid to urolithin A by Limosilactobacillus fermentum FUA033 through rational medium engineering, while deciphering the molecular orchestration underlying this metabolic process. Transcriptome sequencing technology was utilized to analyze L. fermentum FUA033 before and after ellagic acid supplementation in media, and predict the molecular mechanisms involved in the biotransformation of ellagic acid into urolithin A. The optimized medium was determined to be Wilkins-Chalgren Anaerobe Broth (WAM) supplemented with 0.16% trace element solution, 0.16 mmol/L methyl viologen, and 0.21% vitamin solution. The optimization resulted in biotransformation rate of 74.91%, 20.83% improved as compared to the unoptimized condition. Following the addition of ellagic acid, 64 significantly up-regulated genes, and 123 differentially down-regulated genes. GO enrichment analysis demonstrated that the differentially expressed genes were significantly enriched in molecular functions including transporter activity, membrane, and ion transport. The KEGG pathway enrichment analysis demonstrated significant alterations in oxidative phosphorylation (P<0.05), with substantial enrichment of functional genes related to metabolic pathways and the biosynthesis of secondary metabolites. Notably, two lactonase-encoding genes exhibited significant upregulation, strongly suggesting their catalytic role in hydrolyzing ellagic acid's lactone rings to generate the urolithin A precursor. These findings establish a mechanistic foundation for microbial biosynthesis of urolithin A through fermentation, while systematically delineating the enzymatic determinants governing the bacterial-mediated biotransformation of ellagic acid into urolithin A.