Immune agonist immunotherapies have excellent anti-tumour activity in numerous cancer models, alone or in combination with other immunotherapies including immune checkpoint inhibitors. However, a major obstacle to the clinical translation of immune agonists is dose-limiting toxicity and in a substantial proportion of patients, serious immune-mediated side effects including cytokine release syndrome and serious liver damage. Excitingly, we have found that the gut microbiota is a critical component in the development of the toxicity induced by anti-CD137 and anti-CD40 immune agonists. Mice depleted of their gut microbiota with antibiotics, or germ-free mice without a natural microbiota, have a significantly reduced cytokine storm (serum TNFa and IL6 were significantly reduced) and were almost completely protected from the liver damage (significantly reduced ALT and liver necrosis) induced by these therapies. Colitis induced by anti-CD40 was also significantly reduced in microbiota depleted mice. Analysis of liver immune cells demonstrated that microbiota depletion significantly reduced monocyte (CD11b+Ly6G-Ly6C+) macrophage (CD11b+Ly6G-F4/80+) and lymphocyte (CD4+, CD8+ T-cells) infiltration and activation (Ki67, CD80, CD86) following anti-CD40 and anti-CD137 treatment. Importantly, despite potently reducing the cytokine storm and liver damage toxicity, depletion of the gut microbiota had a minimal impact on the anti-tumour efficacy of anti-CD40 or anti-CD137 against transplanted subcutaneous breast and colon cancers. These data suggest that targeting the gut microbiota is a novel strategy to overcome the toxicity associated with immune agonists, potentially allowing higher and more effective doses to be used safely in patients.