Background: Gallbladder cancer (GBC) is the seventh most common gastrointestinal cancer. Suppression of autophagy contributes to cell death of gallbladder cancer. Gensenoside Rg3 sensitizes tumor cells to chemotherapeutic agents through autophagy inhibition. However, its role mechanism on the progression of GBC remains vague. The present study is aimed to explore the functional action of Rg3 on GBC progression. Methods: Expression of miR-181b and CREBRF in human gallbladder carcinoma specimen were determined by western blotting and qRT-PCR. Biological character of tumor cells were assessed by FACS, CCK8 and xenograft assays, respectively. Dual luciferase assay was employed to explore the targeting site of miR-181b. Autophagy flux was detected by IF staining. Results: MiR-181b expression was increased, while CREBRF expression was reduced in GBC specimens compared to adjacent normal tissues. Based on Catalogue of Somatic Mutations in Cancer (COSMIC) database (408 GBC samples), there was negative correlation between hsa-miR-181b-5p/-3p and CREBRF which was a direct targeting of miR-181b. miR-181b mimic promoted cell proliferation and autophagy, restrained cell apoptosis by regulating CREBRF/CREB3 pathway. As an anti-tumor agent, gensenoside Rg3 inhibited cell proliferation and tumor growth, while promoted cell apoptosis by inhibiting autophagy. However, exogenous miR-181b blunted Rg3-evoked anti-tumor effect possibly by inhibiting CREBRF/CREB pathway. Conclusion: Collectively, these data indicates that miR-181b possibly mediates the pathologic progression of GBC by CREBRF/CREB3 signaling pathways and impairs anti-tumor effects of Rg3 on GBC development, which suggests that miR-181b might be an key switch in the process of Rg3-mediated tumor cytotoxicity in the progression of GBC.