Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida has been overexpressed in Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 150.7, c = 167.1 A. Native data were collected to 1.8 A resolution by a rotation method at 100 K using an ADSC Quantum 4R CCD detector with synchrotron radiation.