Inflammatory bowel disease patients exhibit dysregulated bile acid (BA) metabolism in the gut, including elevated levels of host and microbial conjugated bile acids (MCBAs). Because of their novelty, the specific mechanisms behind the increase in MCBAs remain unclear. Bile salt hydrolase (BSH) is a key enzyme in microbial BA metabolism, not only hydrolyzing host-conjugated BAs but also possessing N-acyltransferase activity to generate MCBAs. Our previous study showed a significant increase in the Ruminococcus gnavus bsh gene in Crohn’s disease (CD) patients. Phylogenetic and clustering analysis revealed high intraspecies conservation among RgBSHs, in two major distinct clusters (Cluster 1 and Cluster 2) that contained 9 and 126 genomes, respectively. Clinical data revealed that Cluster 2 RgBSH was significantly more abundant in CD patients and showed a strong positive correlation with specific MCBAs (cholic acid- and chenodeoxycholic acid-conjugated). In vitro functional assays validated this distinction: a representative strain carrying the Cluster 2 bsh gene showed weak hydrolytic activity but significantly enhanced reconjugation activity. Conversely, the Cluster 1 strain exhibited weaker synthesis of MCBAs but slightly higher hydrolysis. Purified enzyme experiments further confirmed that both RgBSH clusters possessed broad N-acyltransferase and hydrolytic activities against multiple BAs. These findings suggest that specific RgBSH subtypes, particularly Cluster 2 RgBSH, may drive the dysregulated BAs metabolism in inflammatory bowel disease (IBD) patients through their unique enzymatic functions, offering new insights into the contribution of R. gnavus to IBD pathogenesis.