ATP-sensitive K+ channels (KATP channels) play an important role in glucose homeostasis. A single nucleotide polymorphism (SNP) in the Kir6.2 subunit causes a point mutation of Glu23 to lysine and reduces the ATP sensitivity of pancreatic KATP channels. The SNP found in 58% of Caucasians accounts for 15% of type 2 diabetes. Here we show evidence for dysregulations of muscular KATP channels with the E23K variation. We were particularly interested in the channel modulation by intracellular protons, as pH changes widely and frequently in skeletal muscles. Surprisingly, we found that the defect of the E23K variant was more related to pH than ATP. A level of intracellular acidification seen during exercise not only activated the E23K channel more readily than the wild type, but also relieved the channel inhibition by ATP, leading to a vast increase in the channel open-state probability by approximately sevenfold at pH 6.8 over the wild-type channel at pH 7.4. Considering the reduction in sarcolemmal excitability, muscle fatigue, and impairment of muscular glucose uptake found previously by genetically disrupting KATP channels, it is likely that the E23K variant in muscular KATP channels affects systemic glucose homeostasis and poses an important risk factor for type 2 diabetes and obesity.