Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. A newly potential therapy – chimeric antigen receptor (CAR) T cell therapy utilizes genetically modified T cells that specifically recognize surface antigen on cancer cells without restriction by major histocompatibility complex (MHC). Mucin 1 (MUC1) is an attractive candidate antigen due to its high expression in CCA cells, and its association with poor prognosis and survival. Since anti-MUC1-CAR T cells have not previously been tested for activity in models of CCA, we set forth to test their utility in this setting. A fourth generation anti-MUC1-CAR construct was engineered to contain anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ. Cultures of anti-MUC1-CAR T cells consisted primarily of cytotoxic (CD8+) T cells (75.13±11.65%, p<0.001). Anti-MUC1-CAR T cells produced increased levels of IFN-γ when exposed to MUC1-positive KKU-100 and KKU-213A CCA cells (31.33±6.02% and 46.5±8.82%, respectively; both p<0.05). Moreover, anti-MUC1-CAR T cells demonstrated specific killing activity against KKU-100 (45.88±7.45%, p<0.05) and KKU-213A cells (66.03±3.14%, p<0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against control immortal cholangiocytes (MMNK-1 cells). These activities of anti-MUC1-CAR T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA.