Nudix hydrolases are a family of proteins that contains the Nudix signature of the characteristic amino-acid sequence Gx5Ex5[UA]xREx2EExGU, where x represents any amino acid and U usually a bulky hydrophobic amino acid, such as Leu, Val, or Ile. They ubiquitously exist in more than 200 species. YmfB, a novel Nudix hydrolase found in Escherichia coli, is a nonspecific nucleoside tri- and di-phosphatase, which atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. In this study, YmfB was cloned, overexpressed, and crystallized. Two different crystals, one belonging to an orthorhombic space group C2221 and the other a monoclinic space group P21, diffracted to 2.7 Å and 2.6 Å resolution, and had unit cell parameters of a = 68.7 Å, b = 283.1 Å, c = 70.4 Å and a = 69.1 Å, b = 70.3 Å, c = 145.6 Å, β = 103.3°, respectively. For the C2221 space group, four or five monomers exist in the asymmetric unit, with a corresponding Vm of 2.48 or 1.99 Å3 Da-1 and a solvent content of 50.5 or 38.2%. For the P21 space group, eight or nine monomers exist in the asymmetric unit, with a corresponding Vm of 2.49 or 2.21 Å3 Da-1 and a solvent content of 50.7 or 44.5%.