Human protein DJ-1 protects neurons from oxidative stress and early onset of Parkinson’s disease via an unknown mechanism. We have recently demonstrated that a small fraction of hexahistidine-tagged human DJ-1 overexpressed in bacterial cells undergo a transient post-translational modification on reactive cysteine (Cys106) forming a stable carboxymethyl adduct upon purification of DJ-1 from the bacterial lysate. Here, we demonstrate that compared to bacterial expression, a much higher proportion of DJ-1 is modified on Cys106 when the protein is overexpressed in methylotrophic yeast. Since methylotrophic yeast oxidizes methanol using molecular oxygen to generate hydrogen peroxide and formaldehyde, DJ-1 is likely to be exposed to a significant oxidative stress, suggesting that the extent of transient post-translational modification may reflect the severity of oxidant stress. To test this hypothesis we have studied whether the extent of the post-translational modification of Cys106 correlates with oxidative stress. We have found that rotenone-induced oxidative stress increases the amount of posttranslationally modified DJ-1 in mammalian cells. The addition of mouse brain cytosol to cell lysate with overexpressed DJ-1 resulted in a reduction of transiently modified DJ-1 suggesting the existence of a pathway that converts modified DJ-1. We conclude that an unknown metabolite reacts with Cys106 of DJ-1 to result in a relatively stable post-translational modification. This modification is different from simple oxidation to sulfinic or sulfonic acids and confers altered binding properties to DJ-1 suggesting that it may serve as a signal for sensing of oxidant stress.