TREX1 is an autonomous 3-prime exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrates that this sequence is required for ubiquitination at multiple lysine residues through a non-canonical ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Co-transfection studies indicate ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutieres syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to TREX1 WT suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated, disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology leading to human autoimmune disease.