STING is a pattern recognition receptor that activates type I interferon and proinflammatory responses in 51 addition to unrelated molecular processes following exposure of DNA to the cytosol. Its pharmacologic 52 stimulation enhances vaccine potency and generates effective antitumor responses but clinical trials evaluating 53 STING agonists have not led to approval for human use. STING activation can occur through ligand 54 engagement of either cytosolic or transmembrane protein domains, processes to which distinct cellular 55 phenotypes are attributed. However, the only transmembrane STING agonist identified is human selective and 56 in vivo testing in conventional models is not feasible. Here we describe synthesis of novel STING agonists 57 efficacious against allelic variants of the protein. We also describe genetically humanized STING mice and 58 demonstrate their suitability as a model to evaluate in vivo responses following exogenous administration of 59 human-selective agonists. Experiments demonstrate that the lead molecule (termed INI3069) functions through 60 binding to the STING transmembrane region and its comparison with conventional agonists reveals significant 61 differences in molecular and immune effects. INI3069 can also enhance antibody responses to co62 administered antigens and antitumor responses. This work both represents the first in vivo examination of the 63 effects of transmembrane STING agonism and demonstrates efficacy of a potential novel vaccine adjuvant and 64 oncological therapeutic.