Distal cholangiocarcinoma is a rare and highly aggressive malignant tumor. The inherent tumor characteristics and growth pattern of cancer cells pose a challenge for diagnosis and treatment. Chemotherapy resistance leads to limited treatment options for patients with advanced cholangiocarcinoma. However, drug resistance studies in cholangiocarcinoma are often limited by the use of preclinical models that do not accurately replicate the essential features of the disease. In this study, we established and characterized a primary multidrug-resistant distal cholangiocarcinoma cell line, CBC3T-6. STR profiling indicated no evidence of cross-contamination. This cell line remains stable during long-term in vitro culture and is characterized by short doubling times and rapid subcutaneous tumor formation in mice. In addition, among the first-line anticancer drugs for cholangiocarcinoma, CBC3T-6 cells showed varying degrees of resistance to gemcitabine, oxaliplatin, cisplatin, and 5-FU. Whole exome sequencing analysis revealed that CBC3T-6 cells contained a variety of potentially pathogenic somatic cell mutations, such as TP53 and KRAS mutations. ABCB1 mutation as a possible therapeutic target for multidrug resistance. In conclusion, CBC3T-6 cells can be used as a useful tool to study the mechanism of cholangiocarcinoma and develop new therapeutic strategies for multidrug resistance.