Oncolytic vaccinia virus (VACV) is a promising candidate for cancer immunotherapy due to its ability to infect a wide range of cancer cells. This review discusses the mechanisms, benefits, and clinical trials of oncolytic VACVs. The safety and efficacy of different viral backbones are explored, as well as the effects of oncolytic VACVs on the tumor microenvironment. The potential combination of oncolytic VACVs with immunotherapy or traditional therapies is also highlighted. The review concludes by addressing prospects and challenges in the field of oncolytic VACVs, with the aim of promoting further research and application in cancer therapy. Oncolytic virotherapy (OVT) uses genetically engineered viruses to replicate within cancer cells and trigger anti-tumor immune responses. OVs can kill cancer cells, remodel the tumor microenvironment, and stimulate long-term anti-tumor immune responses. Despite positive results in cellular and organismal studies, only a few oncolytic viruses are approved for clinical use. VACV has emerged as a potential candidate due to its ability to infect a wide range of cancer cells. VACV is a large DNA prototypic poxvirus that replicates exclusively in the cytoplasm and is fully nonintegrative. It has been used as a smallpox vaccine with relatively low adverse reactions. Recent preclinical results and clinical data about different engineered oncolytic VACVs show its potential for intravenous infusion and tumor therapy via highly and stably expressing many therapeutic genes. VACVs have several advantages in OVT, including exclusive cytoplasmic replication, large genome, natural tumor tropism, rapid and lytic replication cycle, and ability to replicate in hypoxic conditions. Oncolytic VACVs can exert anti-tumor effects via oncolysis and activation of anti-tumor immune responses. The key VACV genes and corresponding oncolytic functions are listed in Table 1. Deletion of those genes can improve the antitumor efficacy through multiple ways such as increasing the tumor selectivity, safety, and anti-tumor immune response. Oncolytic VACVs primarily destroy tumor tissues via three mechanisms: direct oncolysis of tumor cells, disrupting tumor vasculature, and activating anti-tumor immunity. Oncolytic VACVs can selectively replicate in tumor cells and lyse them, while sparing normal cells. The selective oncolysis of oncolytic VACVs mainly lies on the differences between tumor cells and normal cells. On the one hand, various tumor suppressor genes and antiviral signals are significantly downregulated, making it easier for oncolytic VACVs to survive and replicate in the tumor cell. On the other hand, most of oncolytic VACVs are constructed by deletion of some viral genes that overexpressed in tumor cells. Oncolytic VACVs can disrupt tumor vasculature by inducing cytokOncolytic vaccinia virus (VACV) is a promising candidate for cancer immunotherapy due to its ability to infect a wide range of cancer cells. This review discusses the mechanisms, benefits, and clinical trials of oncolytic VACVs. The safety and efficacy of different viral backbones are explored, as well as the effects of oncolytic VACVs on the tumor microenvironment. The potential combination of oncolytic VACVs with immunotherapy or traditional therapies is also highlighted. The review concludes by addressing prospects and challenges in the field of oncolytic VACVs, with the aim of promoting further research and application in cancer therapy. Oncolytic virotherapy (OVT) uses genetically engineered viruses to replicate within cancer cells and trigger anti-tumor immune responses. OVs can kill cancer cells, remodel the tumor microenvironment, and stimulate long-term anti-tumor immune responses. Despite positive results in cellular and organismal studies, only a few oncolytic viruses are approved for clinical use. VACV has emerged as a potential candidate due to its ability to infect a wide range of cancer cells. VACV is a large DNA prototypic poxvirus that replicates exclusively in the cytoplasm and is fully nonintegrative. It has been used as a smallpox vaccine with relatively low adverse reactions. Recent preclinical results and clinical data about different engineered oncolytic VACVs show its potential for intravenous infusion and tumor therapy via highly and stably expressing many therapeutic genes. VACVs have several advantages in OVT, including exclusive cytoplasmic replication, large genome, natural tumor tropism, rapid and lytic replication cycle, and ability to replicate in hypoxic conditions. Oncolytic VACVs can exert anti-tumor effects via oncolysis and activation of anti-tumor immune responses. The key VACV genes and corresponding oncolytic functions are listed in Table 1. Deletion of those genes can improve the antitumor efficacy through multiple ways such as increasing the tumor selectivity, safety, and anti-tumor immune response. Oncolytic VACVs primarily destroy tumor tissues via three mechanisms: direct oncolysis of tumor cells, disrupting tumor vasculature, and activating anti-tumor immunity. Oncolytic VACVs can selectively replicate in tumor cells and lyse them, while sparing normal cells. The selective oncolysis of oncolytic VACVs mainly lies on the differences between tumor cells and normal cells. On the one hand, various tumor suppressor genes and antiviral signals are significantly downregulated, making it easier for oncolytic VACVs to survive and replicate in the tumor cell. On the other hand, most of oncolytic VACVs are constructed by deletion of some viral genes that overexpressed in tumor cells. Oncolytic VACVs can disrupt tumor vasculature by inducing cytok