The genome of the extremely radiation resistant bacterium Deinococcus radiodurans encodes 21 Nudix hydrolases of which only two have been characterized in detail. Here we report the activity and crystal structure for DR_0079, the first Nudix hydrolase observed to have a marked preference for cytosine ribonucleoside 5’-diphosphate (CDP) and cytosine ribonucleoside 5’-triphosphate (CTP). After CDP and CTP the next most preferred substrates for DR_0079, with a relative activity of < 50%, were the corresponding deoxyribose nucleotides, dCDP and dCTP. Hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for three substrates, CDP, IDP, and CTP, and their respective hydrolysis products, CMP + Pi, IMP + Pi, and CMP + PPi. Nucleophilic substitution at the β-phosphorus of CDP and CTP was established, using 31P NMR spectroscopy, by the appearance of an upfield shifted Pi resonance and line-broadened PPi resonance, respectively, when performing the hydrolysis in 40% H218O enriched water. Optimum activity for CDP was at pH 9.0 – 9.5 with the reaction requiring divalent metal cation (Mg2+ > Mn2+ > Co2+). The biochemical data is discussed with reference to the crystal structure for DR_0079 that was determined in the metal-free form at 1.9 Å resolution. The protein contains nine β-strands, three α-helices, and two 310-helices organized into three subdomains; an N-terminal β-sheet, a central Nudix core, and a C-terminal helix-turn-helix motif. As observed for all known structures of Nudix hydrolases, the α-helix of the ‘Nudix box’ is one of two helices that sandwich a ‘four-strand’ mixed β-sheet. To identify residues potentially involved in metal and substrate binding, NMR chemical shift mapping experiments were performed on 15N-labelled DR_0079 with the paramagnetic divalent cation Co2+ and the non-hydrolyzable substrate thymidine-5’-O-(α,β-methylenediphosphate) and the results mapped onto the crystal structure.