A role of glyoxalase I (Gly-I), a detoxifying enzyme, in cell viability of prostate cancer was investigated. Cell extracts obtained from 66 prostate tissue specimens and prostatic cancer PC-3 cells were assayed for Gly-I activity using the spectrophotometric method. Gly-I activity was consistently more than eightfold higher in prostate cancer (CAP) specimens (n = 37) than in non-cancerous (NCP) specimens (n = 29). To understand the importance of such a high Gly-I activity in CAP specimens, the effects of methylglyoxal (MG) on PC-3 cells were examined in vitro. MG, a putative toxic glycolytic metabolite, was capable of inducing severe (> 99%) cell death in 24 h, along with a significant reduction in activities of Gly-I as well as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), a key glycolytic enzyme. However, such severe cell death was effectively (approximately 85%) prevented with N-acetylcysteine (NAC), a precursor of reduced glutathione (GSH) that is an essential cofactor for Gly-I, accompanied by the intact Gly-I and G3PDH activities. Therefore, Gly-I may play a critical detoxifying role in glycolysis to maintain cellular activity and viability of prostatic cancer cells.