Gallbladder cancer (GBC) is one leading cause of cancer-related death worldwide. WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene which can suppress proliferation of a variety of tumors. However, little was known about the relationships between WWOX and gallbladder cancer. In the current study, we intended to investigate the tumor suppressive role of WWOX in gallbladder malignant cells both in vitro and in vivo, and explore the potential mechanism of tumor toxic function of WWOX. Our results have shown that WWOX triggerred apoptosis in GBC cells and increased the expression of P73 and PUMA in cytoplasm. We also have found that Bax has been upregulated after overexpression of WWOX, whereas, Bcl-2 was downregulated by WWOX. To further validate the results in vivo, we evaluated the tumor suppressive role of WWOX in mouse model of gallbladder cancer. The results have shown that the proliferation of the tumor was inhibited after delivery of WWOX, and the expressions of P73 and PUMA were upregulated in target tissues. The mice models administrated with WWOX have shown better prognosis than mice in negative control groups. The results from our study indicated that WWOX could be used as a therapeutic agent in the gene therapy of gallbladder cancer.