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Background Extrachromosomal double-stranded DNA (dsDNA) activates cGAS-STING-dependent innate immune response. In cancer, aberrant incorporation of non-canonical bases such as deoxyuridine (dU) is frequently observed due to antimetabolite therapy and dysregulated cytidine deaminase activity. However, the impact of dU-containing DNA (dU-DNA) on innate immune sensing is yet to be defined.Methods and Results Here, we found that synthetic dsDNA fragment containing dU (dU-DNA) fails to activate type I interferon (IFN) signaling. Mechanistically, dU-DNA does not bind to key DNA sensors cGAS or IFI16 and effectively blocks downstream activation of both cGAS- and STING-mediated pathways. To explore the physiological relevance of this finding, we generated B16F10 melanoma cells deficient in uracil-DNA glycosylase (UNG), the key enzyme responsible for removing uracil from DNA. Building on our previous work showing that ATR inhibition (ATRi) promotes dU incorporation, we treated WT and UNG-deficient B16F10 cells with ATRi and observed significant reduction in type-1 IFN signalling in UNG deficient cells compared to its wild-type counterpart.Conclusions Together, these findings reveal an unexpected immunosuppressive role for dU-DNA by modulating the cGAS-STING pathway. This study has significant importance in the context of cancer-associated DNA damage, repair deficiencies and treatment to anti-metabolite therapy.