Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials.