Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1−/− mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses. Display omitted •NLRX1 inhibits HIV-1 cDNA-induced innate immune response and enhances HIV-1 infection•NLRX1 interacts with the DNA-sensing adaptor STING to disrupt STING-TBK1 signaling•STING deficiency abrogates the enhancement of HIV-1 infection by NLRX1•Nlrx1−/− mice show enhanced innate immunity and are more resistant to DNA viruses Negative regulation of innate immunity is critical to maintain homeostasis, but it can have negative consequences in an infection context. Guo et al. demonstrate that NLRX1 sequesters the DNA-sensing adaptor STING to disrupt STING-TBK1 signaling and inhibit innate immunity to HIV-1 and DNA viruses. Thus, NLRX1 facilitates virus infection.