Background: MicroRNAs are noncoding RNA molecules that posttranscriptionally regulate gene expression. The aim of this study was to determine the role of microRNA-21 in cholangiocarcinomas and its relationship to cholangiocarcinoma RBE cell capacity for invasion and metastasis. Methods: MicroRNA-21 expression was investigated in 41 cases of cholangiocarcinoma samples by in situ hybridization and real-time PCR. Influence on cholangiocarcinoma cell line invasion and metastasis was analyzed with microRNA-21 transfected cells. In addition, regulation of reversion-inducing-cysteine-rich protein with kazal motifs (RECK) by microRNA-21 was elucidated to identify mechanisms. Results: In situ hybridization and real-time quantitative PCR results for patients with lymph node metastasis or perineural invasion showed significantly high expression of microRNA-21 (P<0.05). There was a dramatic decrease in cholangiocarcinoma cell line invasion and metastasis ability after microRNA-21 knockdown (P<0.05). However, overexpression significantly increased invasion and metastasis (P<0.05). Real-time PCR and Western-blot analysis showed that microRNA-21 could potentially inhibit RECK expression in RBE cells. Survival analysis showed that patients with higher expression levels of microRNA-21 more often had a poor prognosis (P<0.05). Conclusions: MicroRNA-21 may play an important role in cholangiocarcinoma invasion and metastasis, suggesting that MicroRNA-21 should be further evaluated as a biomarker for predicting cholangiocarcinoma prognosis.