Cyclic GMP–AMP (cGAMP) is a second messenger that activates the stimulator of interferon genes (STING) innate immune pathway to induce the expression of type I IFNs and other cytokines. Pharmacologic activation of STING is considered a potent therapeutic strategy in cancer. In this study, we used a cell-based phenotypic screen and identified podophyllotoxin (podofilox), a microtubule destabilizer, as a robust enhancer of the cGAMP–STING signaling pathway. We found that podofilox enhanced the cGAMP-mediated immune response by increasing STING-containing membrane puncta and the extent of STING oligomerization. Furthermore, podofilox changed the trafficking pattern of STING and delayed trafficking-mediated STING degradation. Importantly, the combination of cGAMP and podofilox had profound antitumor effects on mice by activating the immune response through host STING signaling. Together, these data provide insights into the regulation of cGAMP–STING pathway activation and demonstrate what we believe to be a novel approach for modulating this pathway and thereby promoting antitumor immunity.