Hydrogen sulfide, an important gasotransmitter, has been implicated in the pathogenesis of diabetes. Increased methylglyoxal (MG) level is linked to the development of type 2 diabetes and hypertension. As a member of the reactive carbonyl species, MG is formed mainly through the nonenzymatic conversion of triosephosphates, such as dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P). The triosephosphate pool, in turn, is regulated by cellular levels of glucose and fructose. The present study investigated whether MG level was altered in mice with cystathionine r-lyase knockout (CSE-KO) and its underlying mechanisms. We measured plasma and renal MG levels in both CSE-KO and wild type (WT) mice at different age groups (6–22weeks). We also evaluated the role of fructose-1,6-bisphosphatase (FBPase) and related signaling pathway in the regulation of MG formation. We observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6–8, 14–16, and 20–22week-old) of the CSE-KO mice. Renal MG, DHAP and GA3P were increased, whereas renal FBPase activity and its mRNA levels were decreased in CSE-KO mice. Decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate, in renal extracts from CSE-KO mice. These data indicate an important role of hydrogen sulfide in the regulation of glucose metabolism and MG generation.