Objective To investigate the specific cytotoxicity of MUC1-specific chimeric antigen receptor (CAR)-engineered Jurkat T cells against hepatocellular carcinoma (HCC) cells. Methods The expression cassettes of both the first and the third generation MUC1-specific CAR gene (i.e. MUC1-CAR and G3MUC1-CAR) were constructed and cloned into lentivirus transfer plasmids, respectively. Then the obtained recombinant lentiviruses carrying the MUC1-specific CAR gene and hrGFP reporter genes were used to infect Jurkat T cells in vitro to establish MUC1-sepcific CAR-engineered Jurkat cells (i.e. CAR-T cells). Subsequently, the assays of CCK-8, ELISA, and LDH were used to detect the cell proliferation, IL-2 secretion and killing effect of the CAR-T cells, respectively. Results We successfully constructed the expression cassettes of MUC1-sepcific CAR gene and the corresponding recombinant lentivirus, established the MUC1-specific CAR-engineered Jurkat T cells. Both types of MUC1-specific CARs-engineered Jurkat T cells could recognize MUC1 molecule and specifically kill MUC1 over-expressed HCC cells while left normal hepatic cells almost undamaged. In addition, the cell proliferation, the secretion of IL-2 and killing effect of the G3MUC1-CAR-engineered Jurkat T cells was significantly superior to the MUC1-CAR-engineered counterpart(P<0.05 or 0.01). Conclusion The MUC1-sepcific CAR-engineered Jurkat T cells can specifically kill MUC1 over-expressed HCC cells.