The cytokine response requires tight regulation of gene and protein expression to ensure no excessive production or prolonged activity of cytokines occurring in acute and chronic inflammation. Negative control mechanisms exist and include both transcriptional and post-transcriptional processes. Among post-transcriptional mechanisms control of cytokine mRNA stability by AU-rich elements which are cis-acting elements in the 3¢UTR. Ribonuclease L (RNase L) is a part of the IFN system and possesses strong activity against viral mRNAs but has also been proposed as a negative feedback player in IFN response targeting specific cellular mRNAs. Also, Type-I IFN is known to have anti-inflammatory action and inhibits the NF-κB pathway, a key element in negative control of gene expression of pro-inflammatory cytokines. In this work, we identified a novel repressor-like protein, the pyrimidine 5'-nucleotidase (NT5C3A) which is an enzyme of nucleotide catabolism. NT5C3A acted as a negative player of interferon and cytokine expression. Specifically, NT5C3A as an intracellular IFN-stimulated gene (ISG) limits inflammatory cytokine production by a cascade of events that involve IRF-1, SIRT1, SIRT6, histone deacetylation, and targeting NFκB interaction with the promoter. Our studies here suggest a novel role for NT5C3A as an anti-inflammatory mediator by inhibiting cytokine expression in a process that involves NF-κB inhibition and NAD+-dependent chromatin modifications during IFN and the cytokine response.