Hepatocellular carcinoma (HCC) exhibits highly invasive and aggressive metastatic characteristics. CXCR3B is one of the three isoforms of the chemokine receptor CXCR3 and has been reported to exert biological effects opposite to those of CXCR3A, including the inhibition of proliferation and angiogenesis and the promotion of apoptosis in cancer cells. Oncolytic viruses (OVs) are viruses that replicate and produce viral particles selectively in tumor cells, lysing them and transforming cold tumors into hot tumors. In this study, we engineered a CXCR3B-expressing oncolytic adenovirus (Ad-TCXCR3B) and evaluated its tumor-suppressive effects in SK-Hep1 HCC cells. We demonstrated that Ad-TCXCR3B exhibited cancer-cell-selective cytotoxicity at various multiplicities of infection (MOIs) and induced apoptosis as confirmed by flow cytometry. Furthermore, Ad-TCXCR3B-mediated CXCR3B overexpression reduced the protein expression of epithelial-to-mesenchymal transition (EMT) markers, including N-cadherin and vimentin, which was associated with decreased invasion and migration of SK-Hep1 cells.