Vaccinia virus (VV), an enveloped DNA virus from the poxvirus family previously played a crucial role in one of the greatest achievements in medicine—the eradication of smallpox. It continues to be used in immunotherapy for prevention of infectious diseases as well as treatment of cancer. Replication-selective oncolytic viruses (RSOVs) have been developed as a new class of cancer therapeutic for the treatment of cancer resistant to conventional therapies. VVs have several inherent features that make them particularly suitable for development as oncolytic agents for cancer treatment. Different VV strains have been used in different areas of the world for mass vaccination. In this chapter, we focus on the potential of the Lister strain (VVL) as a cancer therapeutic agent. The biological features of VVs as RSOVs, cancer selectivity, antitumor efficacy, safety, and future directions are discussed.