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Methylglyoxal (MG) is a cytotoxic compound often produced as a side product of metabolic processes such as glycolysis, lipid peroxidation, and photosynthesis. MG is mainly scavenged by the glyoxalase system, a two-step pathway, in which the coordinate activity of GLYI and GLYII transforms it into D-lactate, releasing GSH. In <i>Arabidopsis thaliana</i>, a member of the GLYI family named GLYI4 has been recently characterized. In <i>glyI4</i> mutant plants, a general stress phenotype characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness was observed. In order to shed some light on the impact of <i>gly4</i> loss-of-function on plant metabolism, we applied a high resolution mass spectrometry-based metabolomic approach to Arabidopsis Col-8 wild type and <i>glyI4</i> mutant plants. A compound library containing a total of 70 metabolites, differentially synthesized in <i>glyI4</i> compared to Col-8, was obtained. Pathway analysis of the identified compounds showed that the upregulated pathways are mainly involved in redox reactions and cellular energy maintenance, and those downregulated in plant defense and growth. These results improved our understanding of the impacts of <i>glyI4</i> loss-of-function on the general reprogramming of the plant’s metabolic landscape as a strategy for surviving under adverse physiological conditions.