Context: GLUT4 is the predominant glucose transporter isoform expressed in fat and muscle. In GLUT4 null mice, insulin-stimulated glucose uptake into muscle was diminished but not eliminated, suggesting that another insulin-sensitive system was present.Objective: This study was intended to determine whether insulin caused GLUT12 translocation in muscle.Design: Six normal volunteers had muscle biopsies before and after euglycemic insulin infusions.Setting: Infusions and biopsies were performed in an outpatient clinic.Participants: Subjects were nonobese, young adults with no family history of diabetes.Main Outcome Measures: GLUT12, GLUT4, and GLUT1 proteins were quantified in muscle biopsy fractions. Cultured myoblasts were used to determine whether GLUT12 translocation was phosphatidyl inositol-3 kinase (PI3-K)-dependent.Intervention. Insulin was infused at 40 mU/m2 · min for 3 h.Results: In human muscle, insulin caused a shift of a portion of GLUT12 from intracellular low-density microsomes to the plasma membrane (PM) fraction (17% in PM at baseline, 38% in PM after insulin). Insulin increased GLUT4 in PM from 13 to 42%. GLUT1 was predominantly in the PM fractions at baseline and did not change significantly after insulin. L6 myoblasts in culture also expressed and translocated GLUT12 in response to insulin, but inhibiting PI3-K prevented the translocation of GLUT12 and GLUT4.Conclusions: Insulin causes GLUT12 to translocate from an intracellular location to the plasma membrane in normal human skeletal muscle. Translocation of GLUT12 in cultured myoblasts was dependent on activation of PI3-K. GLUT12 may have evolutionarily preceded GLUT4 and now provides redundancy to the dominant GLUT4 system in muscle.Insulin induces sequestered GLUT12 to move to the muscle cell surface.