Abstract The efficacy of cancer immunotherapies can be significantly limited by the immune suppressive soluble factors and cell types characterizing the tumor microenvironment. Strategies combining anti-immune suppressive therapies and cancer vaccines need to be evaluated. In the present study, a novel combinatorial strategy was assessed in a mouse model using an aggressive therapeutic setting based on sub-cutaneous ectopic implantation of B16 melanoma cells. The anti-immune suppressive therapy included a novel mix of chemotherapy agents, administered in a metronomic fashion (MCT), and an anti-PD-1 checkpoint inhibitor (CI). The vaccine (VAC) was a multi-peptide cocktail, including melanoma-specific tumor-associated antigens. The newly designed strategy was shown to be safe, well tolerated and highly effective. Animals treated with MCT or CI showed a remarkable delay in tumor growth and prolonged survival as compared to control group. Such effect was significantly enhanced by the combination with the multi-peptide vaccine. In particular, the multi-pronged MCT+CI+VAC combination resulted in a remarkable inhibition of tumor growth in 5/6 animals. The anti-tumor effect was associated with a strong T cell immune response to vaccine peptides as well as significant intra-tumor T cell infiltration associated with reduction in Tregs. Overall, the results showed that the combination of anti-immune suppressive therapy and vaccine was able to strongly potentiate the anti-tumor effects. The described approach is highly promising to significantly enhance the anti-tumor effects induced by a therapeutic cancer vaccine. Citation Format: Maria Tagliamonte, Annacarmen Petrizzo, Angela Mauriello, Antonio Luciano, Domenica Rea, Antonio Barbieri, Claudio Arra, Piera Maiolino, Maria Lina Tornesello, Gennaro Ciliberto, Franco M. Buonaguro, Luigi Buonaguro. Inhibition of tumor growth by combination of metronomic chemotherapy and checkpoint inhibitor with a cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A045.