Acetaminophen (APAP) overdose causes acute liver injury. Our previous study showed that gut microbiota modulates host susceptibility to APAP hepatotoxicity and identified a gut bacterial metabolite phenylpropionic acid (PPA) that alleviates APAP hepatotoxicity in mice. PPA levels in mouse cecum contents exhibit an excellent correlation to those in cardiac serum; inoculation of a PPA-producing gut bacterium Clostridium sporogenes into the mice with minimal cecal and systemic PPA levels significantly increased systemic PPA levels. These results strongly support the notion that gut bacterial PPA production governs PPA systemic exposure in mice. The current study aims to characterize the genetic/biochemical determinants responsible for differential PPA levels in the mouse gut. Our hypothesis is that PPA production in gut bacteria is governed via two previously reported pathways for PPA production from L-phenylalanine (L-Phe): (1) L-Phe reductive pathway catalyzed by enzymes encoded by the fld gene cluster (called Fld pathway; reported in only four different gut bacterial species) and (2) L-Phe ammonia-lyase (PAL) pathway (never reported in gut bacteria to date). LC-MS/MS analysis of mouse cecal samples revealed that L-Phe levels were not correlated with PPA levels, ruling out the role of substrate availability in differential cecal PPA levels. To examine the relative contribution of Fld and PAL pathways to PPA production in the mouse cecum, d8-L-Phe was incubated with mouse cecal bacteria, and the levels of d6- and d7-PPA (products of Fld and PAL pathways, respectively) was measured using LC-MS/MS. As a control, C. sporogenes wild-type and a mutant strain of fldC (encoding an essential subunit within the Fld pathway) were included. Significantly impaired production of d6-labeled PPA was observed in C. sporogenes fldC mutant as expected. The majority of PPA produced from mouse cecal bacteria corresponded to d6-PPA, indicating that Fld pathway is mainly responsible for PPA production under our test conditions. Interestingly, none of the previously reported fldC genes was detected in the mouse cecum by semi-quantitative PCR, suggesting the presence of gut bacteria harboring as-yet-unidentified Fld pathway in mouse cecum. Altogether, we demonstrated the Fld pathway as the major gut bacterial pathway that metabolizes L-Phe to PPA, a gut bacterial metabolite that confers protection against APAP-induced hepatotoxicity. Gut bacteria harboring unknown Fld pathways for PPA production and the role of Fld pathway in PPA cecal levels in vivo remain to be investigated.