Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-D-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-shaped form exhibited diffraction to about 2.0 A resolution. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 A and three dimers of the enzyme per asymmetric unit.