Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.IMPORTANCE Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.