Abstract Biallelic mutations of three prime repair exonuclease 1 (TREX1) cause the lupus-like disease Aicardi–Goutières syndrome in which accumulation of a yet unknown endogenous DNA substrate of TREX1 triggers a cyclic GMP–AMP synthase-dependent type I IFN response and systemic autoimmunity. Products of reverse transcription originating from endogenous retroelements have been suggested to be a major substrate for TREX1, and reverse transcriptase inhibitors (RTIs) were proposed as a therapeutic option in autoimmunity ensuing from defects of TREX1. In this study, we treated Trex1−/− mice with RTIs. The serum RTI levels reached were sufficient to block retrotransposition of endogenous retroelements. However, the treatment did not reduce the spontaneous type I IFN response and did not ameliorate lethal inflammation. Furthermore, long interspersed nuclear elements 1 retrotransposition was not enhanced in the absence of Trex1. Our data do not support the concept of retroelement-derived cDNA as key triggers of systemic autoimmunity in Trex1-deficient humans and mice and motivate the continuing search for the pathogenic IFN-inducing Trex1 substrate.