Tumor cells employ mechanisms to evade anti-tumor immune responses that can include the downregulation or silencing of cytosolic nucleic acid sensing pathway components to dampen Type I IFN signaling. It has been documented that genomic instability and mitotic stress in malignant cells can lead to aberrant cytosolic DNA accumulation and activation of the cGAS-STING pathway. A key negative regulator of cytosolic DNA is the DNA exonuclease TREX1. TREX1 assists in removing DNA from the cytosol, in turn reducing cGAS-STING mediated type I IFN induction. Not surprisingly, TREX1 is overexpressed in several tumor types when compared to normal tissue. The functional importance of TREX1 in cytoplasmic nucleic acid surveillance is evidenced by inactivating TREX1 mutations having an association with Type I inteferonopathies such as Aicardi-Goutières syndrome and SLE. Specifically, most of these mutations impact the enzymatic function of TREX1, underscoring the importance of its exonuclease function. To directly assess TREX1 function in cancer, we inactivated TREX1 in a panel of tumor cell lines. Loss of TREX1 resulted in reduced proliferation and increased expression of the Type I IFN-stimulated gene CXCL10. This finding was validated with CRISPR knockout of TREX1, which also led to the activation of IRF3 and a Type I IFN associated gene signature. To characterize the role of TREX1 function in curbing anti-tumor immunity in vivo, we inactivated Trex1 in the murine syngeneic B16F10 melanoma model. TREX1 knockout together with PD1 blockade resulted in slower tumor growth when compared to PD1 blockade alone. This was accompanied by an increase in CD8+ T and NK cell infiltration in tumors. In addition, gene expression profiling of whole tumors revealed that TREX1 loss resulted in increased type I IFN signaling within the tumor microenvironment. In summary, our data strongly suggest a critical role for TREX1 in suppressing anti-tumor immunity and indicate it would be an attractive target for therapeutic intervention. Citation Format: Haritha Mathsyaraja, Benjamin Wolf, Chari Cortez, Adam Kashishian, Alison Karst, Madelyn Cueva, Min Wang, Kathleen S. Keegan, Jennifer Cain. Tumor cell intrinsic inactivation of TREX1 increases type I IFN signaling and immune cell recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2079.