Extrahepatic Cholangiocarcinoma (EHCC) is one of the uncommon malignancies in the digestive system which is characterized by a poor prognosis. Aberrations of miRNAs have been shown involved in the progression of this disease. In this study, we evaluated the expression and effects of miR-34a on EHCC. miR-34a expression levels were detected in EHCC tissues, adjacent non-tumor tissues, normal bile duct (NBD) specimens of patients and cholangiocarcinoma (CC) cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Relationships between miR-34a with clinical characteristics of EHCC patients were further analyzed. Computational search, functional luciferase assay and western blot were further used to demonstrate the downstream target of miR-34a in CC cells. Immunohistochemistry was carried on to identify the downstream target gene of miR-34a in EHCC patients. Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target. miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients. Smad4 was over-expressed in most of the EHCC patients and was further demonstrated as one of the downstream targets of miR-34a, which was involved in the progression of EHCC. Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro. Taken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.