Silibinin is widely utilized drug in various cancer treatments, though its application in cholangiocarcinoma has not yet been explored. For the first time, we evaluated the anticancer potential and underlying molecular mechanism of silibinin in treatment of cholangiocarcinoma treatment. HuCCT-1 and CCLP-1 cells were chosen to be an study model and were exposed to various concentrations of silibinin for indicated times. Cell viability was evaluated by the cell counting kit-8 (CCK-8) assay and half maximal inhibitory (IC50) concentrations were calculated. Cell proliferation capacity was determined through the use of colony formation and 5-Ethynyl-2'- deoxyuridine (EdU) assays. Cell apoptosis and cycle arrest were assessed by Live/Dead staining assay and flow cytometry (FCM). The protein levels of extracellular regulated protein kinases (ERK)/mitochondrial apoptotic pathway were evaluated through western blotting (WB). Mitochondrial membrane potential changes were determined 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1). A cholangiocarcinoma cell line xenograft model was used to assess the anti-tumor activity of silibinin . Inhibition of the ERK protein by silibinin led to a significant decrease in mitochondrial membrane potential, which, in turn, caused Cytochrome C to be released from the mitochondria. The activation of downstream apoptotic pathways led to apoptosis of cholangiocarcinoma cells. In general, silibinin inhibited the growth of cholangiocarcinoma cell line xenograft tumors. Silibinin is able to inhibit cholangiocarcinoma through the ERK/mitochondrial apoptotic pathway, which makes silibinin a potential anti-tumor drug candidate for cholangiocarcinoma treatment.