Objective: Hydroxyacylglutathione hydrolase (aka as GLO-2) is a component of the glyoxalase pathway involved in the detoxification of the reactive oxoaldehydes, glyoxal and methylglyoxal. These reactive metabolites have been linked to a variety of pathological conditions, including diabetes, cancer and heart disease and may be involved in the aging process. The objective of this study was to generate a mouse model deficient in GLO-2 to provide insight into the function of GLO-2 and to determine if it is potentially linked to endogenous oxalate synthesis which could influence urinary oxalate excretion. Methods: A GLO-2 knock out mouse was generated using CRISPR/Cas 9 techniques. Tissue and 24-h urine samples were collected under baseline conditions from adult male and female animals for biochemical analyses, including chromatographic measurement of glycolate, oxalate, glyoxal, methylglyoxal, D-lactate, ascorbic acid and glutathione levels. Results: The GLO-2 KO animals developed normally and there were no changes in 24-h urinary oxalate excretion, liver levels of methylglyoxal, glyoxal, ascorbic acid and glutathione, or plasma d-lactate levels. GLO-2 deficient males had lower plasma glycolate levels than wild type males while this relationship was not observed in females. Conclusions: The lack of a unique phenotype in a GLO-2 KO mouse model under baseline conditions is consistent with recent evidence, suggesting a functional glyoxalase pathway is not required for optimal health. A lower plasma glycolate in male GLO-2 KO animals suggests glyoxal production may be a significant contributor to circulating glycolate levels, but not to endogenous oxalate synthesis.