Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K(+) (K(ATP)) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3'-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3'-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular K(ATP) channels, and impairment of arterial function.