Hexafluoropropylene oxide trimer acid (HFPO-TA) is reported to have hepatotoxicity, lipotoxicity, and cytotoxicity. In this study, the toxicological effects of HFPO-TA on mitochondrial function and biogenesis were studied. Mice were exposed to drinking water which contained either 2, 20, or 200 μg/L HFPO-TA. Results showed exposure to HFPO-TA induced disadvantageous physiological changes in mice, including increases in liver weight, altered cell morphology, and inflammatory responses. Specifically, exposure to 200 μg/L HFPO-TA increased mitochondria number, relative mitochondrial DNA (mtDNA) content, and mRNA levels of mitochondrial genes encoded by mtDNA. Significant increases in TFAM mRNA and protein levels were also observed. Liver metabolome analysis also showed exposure to 200 μg/L HFPO-TA further enhanced increases in metabolites and altered metabolic pathways that correlated with mitochondrial function, especially the production of ATP. HFPO-TA exposure increased protein expression of mitochondrial complex I-V, and the activities of key enzymes involved in TCA cycle (α-ketoglutarate dehydrogenase, citrate synthase, and succinate dehydrogenase). Furthermore, exposure to 200 μg/L HFPO-TA significantly up-regulating mRNA and protein levels of Opa1, Mfn1, Mfn2, Fis1, and Mff, but did not change Drp1. These findings suggest HFPO-TA could have detrimental effects on health of animals, particularly it was associated with disrupted mitochondrial energy metabolism.