Metabolic reprogramming is a hallmark of cancer. Aberrantly-activated metabolic enzymes fuel tumor progression through multiple mechanisms, including tumor cell proliferation, metastasis and drug resistance. Elucidating the regulatory mechanism of metabolic enzymes in cancer is thus of vital importance, which will pave a way to novel anti-tumor therapies. The first three steps of de novo pyrimidine synthesis are mediated by a multi-functional enzyme known as carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD). Our lab have identified CAD as a protein deamidase that deamidates the NF-κB transcriptional factor RelA, shunting RelA from mediating the transcription of inflammatory cytokines to glycolytic enzymes. CAD thereby acts as a cellular metabolic node that fuels cancer cell proliferation by mediating pyrimidine synthesis and aerobic glycolysis. Indeed, CAD has been characterized as a critical oncogenic factor in a variety of malignancies. However, whether and how CAD is regulated by innate immune response remain unexplored. In this study, we report that innate immune activation dramatically impairs CAD enzymatic activity in colon cancer cells. Mechanistically, CAD is phosphorylated at multiple residues by two key innate immune protein kinases, TANK-binding kinase-1 (TBK1) and inhibitor of NF-κB kinase-beta (IKKbeta). TBK1 / IKKbeta-mediated phosphorylation significantly attenuates both pyrimidine synthetase and protein deamidase activity of CAD, subsequently suppressing colon cancer cell metabolic reprogramming and proliferation. Furthermore, TBK1 / IKKbeta-mediated CAD phoshorylation results in pyrimidine depletion and an imbalance in cellular nucleotide pool, which sensitizes colon cancer cells to DNA damage agents. Pharmacological activation of innate immune signaling by stimulator of interferon gene (STING) agonist can thereby inhibit intratumoral CAD activity and boost chemotherapy agents-induced DNA damage, highlighting a novel combination therapy. Besides, in a panel of cancer cell lines in which TBK1 acts as an oncogene, TBK1 is switched to activate CAD via growth factor signaling pathway, implicating context-dependent roles of TBK1 in regulating CAD. In summary, our study reveals an unprecedented crosstalk that places pyrimidine synthesis as a downstream ramification of innate immune activation. These findings provide fresh insights into how innate immune signaling reshapes tumor metabolic landscape, and offer a mechanistic rationale for combining STING agonists with chemotherapy as a highly potent approach to improve cancer treatment.