Methylglyoxal production is increased in diabetes. Methylglyoxal is efficiently detoxified by enzyme glyoxalase 1 (GLO1). The aim was to study the effect of diabetic and CKD milieu on (a) GLO1 gene expression in peripheral blood mononuclear cells; (b) GLO1 protein levels in whole blood; and (c) GLO1 activity in RBCs in vivo in diabetic vs. non-diabetic subjects with normal or slightly reduced vs. considerably reduced renal function (CKD1-2 vs. CKD3-4). A total of 83 subjects were included in the study. Gene expression was measured using real-time PCR, and protein levels were quantified using Western blotting. Erythrocyte GLO1 activity was measured spectrophotometrically. GLO1 gene expression was significantly higher in subjects with CKD1-2 compared to CKD3-4. GLO1 protein level was lower in diabetics than in non-diabetics. GLO1 activity in RBCs differed between the four groups being significantly higher in diabetics with CKD1-2 vs. healthy subjects and vs. nondiabeticsfig with CKD3-4. GLO1 activity was significantly higher in diabetics compared to nondiabetics. In conclusion, both diabetes and CKD affects the glyoxalase system. It appears that CKD in advanced stages has prevailing and suppressive effects compared to hyperglycaemia. CKD decreases GLO1 gene expression and protein levels (together with diabetes) without concomitant changes of GLO1 activity.