Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in the glycolytic pathway. In addition to its conventional metabolic role, GAPDH has been identified to possess diverse cellular functions. In this study, glyceraldehyde-3-phosphate dehydrogenase 3, the third isoform of GAPDH from Saccharomyces cerevisiae, was cloned, expressed, purified and crystallized. The crystals belonged to space group I4(1)22, with unit-cell parameters a = b = 116.13, c = 119.21 Å. X-ray diffraction data were collected to a resolution of 2.6 Å. The structure was solved by molecular replacement and refinement is in progress.