2,2′,3,5’,6-Pentachlorobiphenyl (PCB95) is known as a persistent pollutant that was found in eggs in China. PCB 95 can be metabolized into OH-PCB95 and MeO-PCB95 in liver microsomes. However, the toxicity and its mechanism of PCB95 or its metabolites have been little studied on laying hens. Herein, chicken embryo liver cells of laying hens were selected and treated with different levels of PCB95 and its two metabolites, and the EC₅₀ of PCB95, OH-PCB95, MeO-PCB95 was 80.85, 4.81 and 107.04 μg/mL respectively, indicating that OH-PCB95 is much more cytotoxic than PCB95 or MeO-PCB95. Targeted metabolomics was further used to study the effects of the parent compound and its metabolites on cell metabolism. The results showed that four primary types of glycerophospholipids were down-regulated after exposure to PCB95 and its metabolites, especially PE and PS (60% more than the control for PCB95, 40% for OH-PCB95, and less than 40% for MeO-PCB95). KEGG pathway analysis based on amino acid metabolism showed that PCB95 may mainly interfere with the amino acids involved in immune regulation (phenylalanine and tyrosine), and OH-PCB95 may be associated with genetic disoders (cysteine, methionine and purine metabolism). However, the metabolic pathways induced by MeO-PCB95 are quite different from those induced by PCB95 and OH-PCB95, affecting mainly D-glutamine and D-glutamate metabolism, alanine and glutamate metabolism, and arginine and proline metabolism; these pathways mainly regulate the elimination of excess purines and are involved in the synthesis of the amino acids required by cells. These results showed that OH-PCB95 has the highest toxicity on chicken embryo liver cells and MeO-PCB95 could be a detoxification product of PCB95 and OH-PCB95. This study contributes to the understanding of the different effects of PCB95 and its metabolites on cellular metabolism, and the data are helpful in evaluating the hepatotoxic effects of these compounds.