Abstract The tumor immunosuppressive microenvironment represents a major obstacle to an effective tumor-specific cellular immune response. We have previously shown that a novel multi-drug chemotherapy administered in a metronomic fashion was able to increase immune response to peptides. The chemotherapy consisted of a cocktail including taxanes (paclitaxel and docetaxel) and alkylating (cyclophosphamide) agents. The newly designed strategy was shown to be safe, well tolerated and significantly efficacious (Tagliamonte et al., CII 2015). In the present study, the effect on the immunosuppressive tumor microenvironment by the same multi-drug cocktail was evaluated in a mouse model upon sub-cutaneous ectopic implantation of B16 melanoma cells. Treated animals showed a remarkable delay in tumor growth and prolonged survival as compared to control group. Such an effect was directly correlated with CD4+ T cell reduction and CD8+ T cell increase. A significant reduction in the percentage of both CD25+FoxP3+ and CD25+CD127low regulatory T cell population was found in the spleens as well as in the tumor lesions. An intrinsic CD8+ T cell response specific to B16 naturally expressed Trp2 TAA was observed. The same metronomic chemotherapy combined to a vaccine based on mutated antigens, was subsequently shown to increase immune response to vaccine resulting in a significant delay of B16 tumor growth as well as animal survival. The novel multi-drug daily metronomic chemotherapy evaluated in the present study was very effective in counterbalancing the immunosuppressive tumor microenvironment and enhancing vaccine efficacy. Consequently, the intrinsic as well as vaccine-induced anti-tumor T cell immunity could exert its function containing tumor growth. Overall, the described metronomic chemotherapy may represent a promising adjuvant approach to enhance anti-tumor cellular immunity and amplify the biological effects of therapeutic cancer vaccines. Citation Format: Maria Tagliamonte, Annacarmen Petrizzo, Maria Lina Tornesello, Antonio Luciano, Domenica Rea, Antonio Barbieri, Claudio Arra, Maria Napolitano, Gennaro Ciliberto, Franco M. Buonaguro, Luigi Buonaguro. A novel multidrug metronomic chemotherapy significantly delays tumor growth in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 742.