Abstract: ［Abstract］Objective To explore the effect and mechanism of fragile site WWOX gene on regulating proliferation of gallbladder cancer cells in vitro. Methods The pcDNA3.0 - WWOX recombinant plasmid which was previous successfully built was transfected to GBC-SD cells and empty carrier by liposome medium. Liposome and GBC-SD were served as the negative control and the blank control， respectively. After 48 hours transfection， inverted microscope was used to observe the changes of gallbladder cancer cells' morphology， MTT and BrdU were used to detect the proliferation level of gallbladder cancer cells，and flow cytometry instrument was used to detect the change of the cell proliferation cycle. Results The results of inverted microscope shown: the number of GBC-SD cells in pcDNA3.0-WWOX group decreased significantly，the suspension cells and cell debris increased，while cells in the vector control，NC and Mock groups were in normal proliferation state. MTT test showed the proliferation levels of GBC-SD cells in pcDNA3.0-WWOX group was lower than those in the control group in 24 h，48 h，72 h， 96 h and 120 h， and the differences were statistically significant（P < 0.05）. The cell proliferation activity in the pcDNA3.0-WWOX group was obviously inhibited over time. BrdU detection results showed the cell proliferation rate of pcDNA3.0 - WWOX group was（0.44±0.03）， while that in the three control groups was（0.78±0.02）， （0.81±0.01）and（0.85±0.01）， respectively. It showed that cell proliferation activity in pcDNA3.0-WWOX group was lower than the control groups， and the difference was statistically significant （P < 0.05）. Cell cycle detection showed the cells increased in G0/G1 phase and decreased in G2/M and S phases of pcDNA3.0-WWOX group. The cell apoptosis rate was significantly higher and the proliferation index was significantly lower than those of the control groups （P < 0.05）. However， there were no significant differences among the three control groups（P > 0.05）. Conclusion The overexpression of WWOX gene in vitro could effectively inhibit the proliferation activity of gallbladder cancer cells. WWOX might participate in the development of the malignant biological behavior of gallbladder cancer cells. It is expected to become a new potential target for the gene therapy to gallbladder cancer.