The present study aimed to clarify the association between ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) expression level and the tumor phenotype and clinicopathological features of extrahepatic bile duct carcinoma. Tissue samples from patients with extrahepatic bile duct carcinoma (54 cases) and patients with normal bile duct epithelia from gallbladder of cholecystitis (20 cases) were collected, and immunohistochemical staining was used to detect the expression levels of EBP50 in these tissues. In addition, small interfering (si)RNA-EBP50 was used to knock down the expression of EBP50 in the QBC939 human cholangiocarcinoma (CC) cell line. The effect of EBP50 expression on QBC939 cell proliferation and migration was analyzed using the Cell Counting kit-8 and wound healing assays, respectively. EBP50 expression was significantly downregulated in CC tissue samples (P<0.01), with low EBP50 expression levels positively correlated with a high pathological stage and a poor differentiation degree (P<0.01 and P<0.001, respectively). EBP50 expression in QBC939 cells was knocked down by ≤80% using siRNA-EBP50, and EBP50 knockdown significantly promoted QBC939 cell proliferation, as compared with the vector control cells (P=0.04). EBP50 knockdown also significantly enhanced the wound healing ability of QBC939 cells (P=0.02). These results demonstrated that EBP50 expression levels are significantly correlated with a malignant phenotype in patients with CC, and decreased expression levels of EBP50 may promote CC cell proliferation and migration. These findings provide insight into novel potential diagnostic and therapeutic approaches for patients with CC.