Polychlorinated biphenyls (PCBs), such as 2,2',3,5',6-pentachlorobiphenyl (PCB95), are persistent organic pollutants associated with adverse health outcomes, including developmental neurotoxicity. PCB95 is a chiral neurotoxic PCB congener atropselectively metabolized to potentially neurotoxic metabolites in vivo. However, the metabolic pathways of most PCB congeners, including PCB95, remain unknown. To address this knowledge gap, we analyzed the intestinal contents of mice exposed to PCB95 to elucidate the PCB95 metabolism pathway and assess if genetic manipulation of hepatic drug-metabolizing enzymes affects PCB95 metabolism. Our study exposed male and female wildtype (WT), Cyp2abfgs-null (KO), and CYP2A6-transgenic/Cyp2abfgs-null (KI) mice orally to 1.0 mg/kg body weight of PCB95. Intestinal content was collected 24 h after PCB administration. aS-PCB95 was enriched in all intestinal content samples, irrespective of sex and genotype. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analyses identified 5 mono- (OH-PCB95) and 4 dihydroxylated PCB (diOH-PCB95) metabolites. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) identified 15 polar hydroxylated, methoxylated, and sulfated PCB95 metabolites, including 3 dechlorinated metabolites. A sex difference in the relative OH-PCB95 levels was observed only for KO in the LC-HRMS analysis. Genotype-dependent differences were observed for female, but not male, mice, with OH-PCB95 levels in female KO (FKO) mice tending to be lower than those in female WT (FWT) and KI (FKI) mice. Based on the GC-MS/MS analysis, these differences are due to the unknown PCB95 metabolites, X1-95 and Y1-95. These findings demonstrate that combining GC-MS/MS analyses and LC-HRMS subject screening of the intestinal content of PCB95-exposed mice can significantly advance our understanding of PCB95 metabolism in vivo.