Skeletal muscle, a primary insulin target tissue, expresses the GLUT5 fructose transporter. Although insulin has no acute effect on GLUT5 expression and function in muscle, we show here that long-term (24 h) insulin treatment of L6 muscle cells induces a dose-dependent increase in GLUT5 protein (by up to two-fold), leading to a concomitant increase in fructose uptake. The increase in GLUT5 expression and function was suppressed by inhibitors of gene transcription and protein synthesis, suggesting that insulin promotes de novo carrier synthesis. Transfection of the GLUT5 gene promoter fused to luciferase into L6 cells revealed that insulin induced a 1.8-fold increase in GLUT5 promoter activity. Our findings indicate that insulin is capable of increasing the abundance and functional activity of GLUT5 in skeletal muscle cells and that this is most likely mediated via activation of the GLUT5 promoter.