The role of metabolites in shaping mammalian microbiotas remains incompletely understood, due in part to limited knowledge of bacterial metabolism and challenges inferring mechanisms from observational data. In the human vaginal microbiota, bacterial communities dominated by Lactobacillus crispatus are associated with optimal health. In contrast, bacterial communities dominated by Lactobacillus iners or by diverse anaerobes are more commonly linked to adverse health outcomes. We found that women with a vaginal microbiota dominated by L. crispatus had high vaginal concentrations of hippurate (a host-derived metabolite) while women with microbiota dominated by L. iners or various anaerobes had lower vaginal hippurate levels. We hypothesized that this pattern was due to L. crispatus’s inability to metabolize hippurate, whereas L. iners and key vaginal anaerobes hydrolyze hippurate via activity of hippuricase (HipO) enzymes. Using a phenotypic assay, we confirmed that L. iners and numerous vaginal anaerobes exhibit hippuricase activity, while L. crispatus and related non-iners Lactobacillus species lack this activity. We used genomic analysis to identify previously uncharacterized candidate hipO genes in multiple species and confirmed their enzymatic function via cloning and heterologous expression. Characterization of hipO regulation and roles in bacterial physiology is ongoing. These findings explain a key observation from vaginal metabolomics analysis and lay the groundwork for further mechanistic studies of how microbial metabolism shapes microbiota dynamics and function.