Background and Aims MiR-200a-3p participates in the regulation of biological processes in a variety of tumors, but it exerts different effects in different tumors (oncogene or tumor suppressor). At present, its role in gallbladder cancer is still unclear. Therefore, this study was conducted to investigate the expression of miR-200a-3p in gallbladder cancer as well as its effects on biological behaviors of gallbladder cancer cells and the mechanism.Methods The expressions of miR-200a-3p in 32 specimens of gallbladder cancer tissue and adjacent tissue as well as in different gallbladder cancer cell lines (GBC-SD, SGC-996, and NOZ) and normal human gallbladder epithelial cell line HGBEC were determined by qRT-PCR method. The GBC-SD and NOZ cell lines were transfected with miR-200a-3p-mimics (miR-200a-3p overexpression group), miR-200a-3p-inhibitors (miR-200a-3p silencing group) and scramble sequences (negative control group) respectively using Lipofectamine? 3000 kit. The proliferation and invasion abilities of the cells were examined by MTT assay and Transwell assay. The downstream target genes of miR-200a-3p were predicted using MiRBD/Targetscan7.2/starBase2.0/miRtarbase website, and were verified by Luciferase experiment. The protein expressions of the target genes and the molecules (E-cadherin and vimentin) associated with epithelial-to-mesenchymal transition (EMT) in above three groups of cells were measured by Western blot analysis.Results The results of qRT-PCR showed that the expression level of miR-200a-3p in gallbladder cancer tissue was lower than that in adjacent tissue, and the expression levels of miR-200a-3p in all gallbladder cancer cell lines were lower than that in normal human gallbladder epithelial cell line HGBEC (all P<0.05). In GBC-SD and NOZ cell lines after transfection, the expressions of miR-200a-3p were significantly increased and the proliferative and invasion abilities were significantly decreased in miR-200a-3p overexpression groups of both cell lines, while the expressions of miR-200a-3p were significantly decreased and the proliferative and invasion abilities were significantly increased in miR-200a-3p silencing groups of both cell lines compared with their respective negative control groups (all P<0.05). Online website prediction showed that there were potential binding sites between miR-200a-3p and Notch2. Luciferase verification experiments showed that miR-200a-3p caused markedly decreased luciferase activity in Notch2 wild-type plasmid pmirGLO-Notch2-3'UT WT, but exerted no effect on the luciferase activity of Notch2 mutant plasmid pmirGLO-Notch2-3'UTR MUT. The results of Western blot analysis showed the E-cadherin protein expressions were increased and the protein expressions of vimentin and Notch2 were decreased in miR-200a-3p overexpression groups of both cell lines, while the opposite changes in the three proteins were observed in miR-200a-3p silencing groups of both cell lines compared with their respective negative control groups (all P<0.05).Conclusion The expression of miR-200a-3p is decreased in gallbladder cancer, and it may play a tumor suppressor role in gallbladder cancer, and the mechanism may be probably associated with its down-regulating Notch2 and thereby inhibiting the EMT process.