AbstractBacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti‐tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro‐inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow‐derived dendritic cells. Co‐immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma‐bearing mice through Th‐1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti‐PD‐1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti‐tumour responses using immunotherapeutic bacterial SyBV.