Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent. PP inhibits several crucial functions of cancer cells, and it has been patented recently as a potential chemotherapeutic drug for various cancers. The current study investigated the ability of PP in anti-proliferative activity and focused on the lipidomic profiles revealing the alteration of specific lipid species in cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability by suppressing mitochondrial membrane potential (MMP) and ATP production, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with multivariate statistical analysis were performed to investigate lipid alteration of PP-induced apoptosis in CCA cells. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. Moreover, the orthogonal partial-least squares for regression analysis revealed that PP-induced MMP dysfunction, leading to inhibition of ATP contents, was significantly associated with triglyceride production, a characteristic of apoptotic cells. These alterations indicated that PP could suppress the MMP function, which caused inhibition of CCA cell viability through apoptotic induction resulting in lipid accumulation in CCA cells. These findings can provide new insight on an anti-cancer mechanism of PP under apoptotic induction ability that could be a promising therapeutic strategy for CCA treatment.