Perfluorooctansulfonate (PFOS) is a persistent organic pollutant that has attracted a great deal of attention due to toxic effects such as its lipid metabolism-disrupting potential. Exposure to PFOS can cause hepatomegaly and lipoatrophy in mice, but the underlying mechanisms are still unknown. Considering that glyceroneogenesis is the essential pathway for balancing the triglyceride (TG) cycle between liver and white adipose tissue (WAT), we speculate that PFOS acts via glyceroneogenesis inhibition to alter TG metabolism in the two tissues. Combining gene expression, protein expression, an enzyme activity assay, and molecular docking analysis, we report here that PFOS can interact with cytosolic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of glyceroneogenesis. Specifically, by repression of PEPCK, PFOS can inhibit the glyceroneogenesis process and thus decrease the glyceroneogenesis-derived glycerol level, leading to a reduced re-esterified TG level and causing atrophy in WAT. Moreover, in PFOS-exposed liver tissue, despite the fact that free glycerol and fatty acids released from WAT were being used for TG synthesis, the export of TG slowed. This eventually resulted in the continuous lipolysis of WAT and accumulation of lipid in the liver. PEPCK can be used as a key biomarker to assess the lipid metabolism disorders induced by other conventional and emerging per- and polyfluoroalkyl substances.