IFN-beta has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-beta expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-beta expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-beta expression. We determined that cGAS is required for IFN-beta expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-beta, coculture of cells depleted for either STING or cGAS rescued IFN-beta expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-beta expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-beta expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.