There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.