Stimulator of interferon genes (STING, also known as MITA, ERIS, or MPYS) is essential for host immune responses triggered by microbial DNAs. However, the regulatory mechanisms underlying STING-mediated signaling are not fully understood. We report here that, upon cytoplasmic DNA stimulation, the endoplasmic reticulum (ER) protein AMFR was recruited to and interacted with STING in an insulin-induced gene 1 (INSIG1)-dependent manner. AMFR and INSIG1, an E3 ubiquitin ligase complex, then catalyzed the K27-linked polyubiquitination of STING. This modification served as an anchoring platform for recruiting TANK-binding kinase 1 (TBK1) and facilitating its translocation to the perinuclear microsomes. Depletion of AMFR or INSIG1 impaired STING-mediated antiviral gene induction. Consistently, myeloid-cell-specific Insig1^−/− mice were more susceptible to herpes simplex virus 1 (HSV-1) infection than wild-type mice. This study uncovers an essential role of the ER proteins AMFR and INSIG1 in innate immunity, revealing an important missing link in the STING signaling pathway.