〔Abstract〕 Objective To study the effect of miR-181 on the migration and invasion of gallbladder cancer cells by targeting tissue inhibitor of metalloproteinase-3(TIMP3). Methods Gallbladder cancer GBC-SD cells were cultured and transfected with miR-181 mimic, NC mimic, TIMP3 siRNA, NC siRNA, recombinant plasmid expressing TIMP3, blank plasmid. The expression of miR-181 was detected by fluorescence quantitative PCR, the expression of TIMP3 was detected by Western blot, the cell migration and invasion activity were detected by Transwell, and miR-18 target TIMP3 was verified by bioinformatics analysis and luciferase reporter gene experiment. Tumorbearing mouse model was established by subcutaneous injection of GBC-SD cells transfected with miR-181 mimic or NC mimic, then the quality of the transplanted tumor and the expression of miR-181, TIMP3 were detected. Results Cell experiments showed that miR-181 mimic could promote cell migration and invasion, inhibit the expression of TIMP3 and luciferase activity of double luciferase reporter gene; TIMP3 siRNA could promote cell migration and invasion; after transfection of recombinant plasmid expressing TIMP3, the effect of miR-181 mimic on cell migration and invasion was weakened.Animal experiments showed that miR-181 mimic could reduce the weight of transplanted tumor and increase the expression of TIMP3. Conclusion miR-181 promotes the migration and invasion of gallbladder cancer cells, and targeting inhibition of TIMP3 is one of the molecular mechanisms.