Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purine de novo synthesis, and its tissue concentrations can be increased, in vivo, by low doses of methotrexate (MTX) through the inhibition of the enzyme AICAR transformylase. We report here the first evidence that, in experimental type 2 diabetes, chronic treatment with low doses of MTX increases skeletal muscle GLUT4 expression and improves metabolic control. MTX (0.5 mg/kg body weight) or vehicle was administered intraperitoneally, once a week for 4 weeks, to genetically diabetic female C57BL/KsJ- 𝑚 + / + 𝐿 𝑒 𝑝 𝑡 𝑑 𝑏 mice ( 𝑑 𝑏 + / 𝑑 𝑏 + ) and their normoglycemic littermates ( 𝑑 𝑏 + / + 𝑚 ). In the 𝑑 𝑏 + / 𝑑 𝑏 + mice, MTX treatment was associated with a ~2-fold increase in skeletal muscle GLUT4 protein concentration and a >4-fold increase in GLUT4 mRNA expression ( 𝑃 < 0 . 0 1 , all), as compared to vehicle-treated mice; no significant differences were noted in controls. MTX treatment was also associated with a significant reduction of glucose and insulin serum concentrations in diabetic mice ( 𝑃 < 0 . 0 0 1 ), and glucose levels only ( 𝑃 < 0 . 0 5 ) in controls. These data indicate a different route to increase skeletal muscle GLUT4 expression, through the potential inhibition of the enzyme AICAR transformylase.