Staphylococcal nuclease and tudor-domain containing 1 (SND1) is an oncogene for a wide variety of cancers, including hepatocellular carcinoma (HCC). SND1 is a multifunctional protein regulating gene expression of proto-oncogenes and tumor suppressor genes, making SND1 a prime target for developing cancer therapeutics. This notion is especially attributed to HCC as most patients are diagnosed in advanced stages and the therapeutic options available for these patients are severely limited. In this study, we evaluated the therapeutic potential of a replication-defective adenovirus vector delivering SND1 shRNA (Ad.SND1sh) to human HCC cell lines, HepG3, HuH-7, and Hep3B. Adenovirus infection in HCC cells was confirmed by Western blotting and immunofluorescence. The efficacy of Ad.SND1sh to knockdown SND1 expression was confirmed via Western blot, qRT-PCR, and immunofluorescence. Ad.SND1sh did not significantly affect proliferation of the three human HCC cells but significantly inhibited their invasive and migratory capacities, as determined by wound healing and Matrigel invasion assays, respectively. As a corollary, Ad.SND1sh treatment resulted in a decrease in mesenchymal markers, such as N-cadherin, Twist, Snail, and Slug, without affecting levels of epithelial marker E-Cadherin, indicating that SND1 knockdown induces mesenchymal conversion in HCC cells. Additionally, reductions in liver cancer stem cell marker CD133 and HCC marker α-fetoprotein (AFP) were observed with SND1 knockdown. HCC cells with aberrant expression of these markers are associated with tumor initiation, recurrence, and multi-drug resistance. Our findings indicate that Ad.SND1sh may potentially be an effective therapy for advanced HCC and needs to be studied further for its clinical application.