Background and Aims Long non-coding RNA MCM3AP-AS1 (MCM3AP-AS1) plays an oncogene function in various tumors such as primary liver cancer, breast cancer and glioblastoma. However, the expression, function and mechanism of MCM3AP-AS1 in cholangiocarcinoma are still poorly understood. Therefore, this study was conducted to observe the expression of MCM3AP-AS1 in cholangiocarcinoma cells and its effect on cell proliferation and invasion, and preliminarily investigate the underlying mechanism.Methods The expressions of MCM3AP-AS1 in cholangiocarcinoma cell lines (CCLP, RBE, 9810, HuCCT1) and human intrahepatic bile duct epithelial cell line (HIBEC) were detected by qRT-PCR. In cholangiocarcinoma cells after transfection with MCM3AP-AS1 siRNA, using cholangiocarcinoma cells transfected with scrambled sequences as negative control, the changes in proliferative and invasion abilities were determined by MTT assay and Transwell assay, and the changes in expressions of the proteins associated with the JAK/STAT3 signaling pathway and epithelial-mesenchymal transition (EMT) process were determined by Western blot. Finally, functional rescue experiment was performed using the JAK/STAT3 pathway agonist leukemia inhibitory factor (LIF) for validation.Results The expressions of MCM3AP-AS1 in all cholangiocarcinoma cell lines were significantly higher than that in HIBEC (all P<0.05). In CCLP cells after transfection with MCM3AP-AS1 siRNA compared with negative control, the proliferative and invasion abilities were significantly decreased (both P<0.05); the expression levels of JAK1/2 and STAT3 showed no significant changes (both P>0.05), but the expression levels of p-JAK1/2 and p-STAT3 were significantly decreased (both P<0.05), and meanwhile, the expression of EMT-related protein E-cadherin was increased and vimentin was decreased (both P<0.05). The results of functional rescue experiment showed that the effects of MCM3AP-AS1 silencing on CCLP cells were all abolished by simultaneous addition of LIF, and all parameters had no significant differences with those in negative control group (all P>0.05).Conclusion The expression MCM3AP-AS1 is up-regulated in cholangiocarcinoma cells, and it promotes the cell proliferation and invasion of the cholangiocarcinoma cells probably through activating the JAK/STAT3 signaling pathway and EMT process.