Advanced glycation end-products (AGEs) are irreversible protein modifications that are strongly associated with aging and disease. Recently, the Parkinsonism-associated protein DJ-1 has been reported to exhibit deglycase activity that erases early glycation intermediates and stable AGEs from proteins. In this work, we use mass spectrometry and western blot to demonstrate that DJ-1 is not a deglycase and cannot remove AGEs from protein or peptide substrates. Instead, our studies revealed that DJ-1 antagonizes glycation through glyoxalase activity that detoxifies the potent glycating agent methylglyoxal (MGO) to lactate. We further show that attenuated glycation in the presence of DJ-1 can be attributed solely to its ability to decrease the available concentration of MGO. Our studies also provide evidence that DJ-1 is allosterically activated by glutathione. Together, this work reveals that although DJ-1 is not a genuine deglycase, it still harbors the ability to prevent AGE formation and can be used as a valuable tool to investigate metabolic stress.