Glyoxalase pathway is the major pathway of methylglyoxal detoxification and is ubiquitously present in all organisms ranging from prokaryotes to eukaryotes. Glyoxalase I (GLYI) and Glyoxalase II (GLYII), the two core enzymes of this pathway work together to neutralize methylglyoxal (MG), a dicarbonyl molecule with detrimental cytotoxicity at higher concentrations. The first step towards the detoxification of MG is catalyzed by GLYI, a metalloenzyme that requires divalent metal ions (either Zn2+ as seen in eukaryotes or Ni2+ as in prokaryotes). However, both Zn2+ and Ni2+ dependent GLYIs have been shown to co-exist in a higher eukaryote i.e. Arabidopsis thaliana. In the present study, we determine the role of both Zn2+ dependent (AtGLYI2) and Ni2+ dependent (AtGLYI3, AtGLYI6) GLYIs from Arabidopsis in salinity stress tolerance. AtGLYI2 overexpressing Arabidopsis plants showed better growth rate while maintaining lower levels of MG under high saline conditions. They were taller with more number of silique formation with respect to their Ni2+ dependent counterparts. Further, lack in germination of Arabidopsis AtGLYI2 mutants in presence of exogenous MG indicates the direct involvement of Zn2+ dependent GLYI in MG detoxification, suggesting Zn2+ dependent GLYI as the main enzyme responsible for MG detoxification and salinity stress tolerance.