Vesicular stomatitis virus has been known as a potent antitumor agent because of its selective replication and lysis of tumor cells and immune-stimulating properties. In response to cellular stress and enhanced metabolism, tumor cells activate autophagy, to provide energy for the cells and preventing tumor destruction. Inhibition of autophagy can increase the therapeutic potential of many antitumor methods. This study aimed to check the efficacy of combined VSV and three-methyl adenine (3-MA) in treating a tumor model in mice. TC-1, a line of C57BL/6 mouse lung cells transformed by HPV-16 E7 and E6 oncoproteins, as well as human Ras, were used for experiments. The viability after treatment with the optimized concentration of 3-MA with or without combination with VSV was assessed by MTT. C57BL/6 male mice were injected with TC-1, and after tumor formation, 3-MA and VSV alone or in combination in two different protocols were injected into tumor mice. Tumor size, tumor-specific CTL response, and apoptosis rate were evaluated. The results showed that 3-MA combined with VSV causes more lethality in tumor cells in vitro. In vivo studies also showed that combined VSV and 3-MA treatment inhibits the progression of TC-1 cancer cells with higher efficiency, especially in daily 3-MA treatment along with four doses of VSV injection with four days' intervals. In addition, the rate of apoptosis and cytotoxic T cells activity in the groups injected with 3-MA and the virus were higher than groups receiving each agent alone. In conclusion, the association of VSV with 3-MA increases its oncolysis activity and subsequently more stimulates the immune system against the tumor. This finding suggests a combinational approach for tumor therapy with therapy. Combining oncolytic VSV with 3-MA as an autophagy inhibitor agent can improve the efficacy of tumor treatment. This combination therapy approach enhances apoptosis in tumors as well as T cell cytotoxicity against tumor cells.