Mouse brain expresses three principal glucose transporters. Glut1 is an endothelial marker and is the principal glucose transporter of the blood-brain barrier. Glut3 and Glut6 are expressed in glial cells and neural cells. A mouse line with a null allele for Glut3 has been developed. The Glut3-/- genotype is intrauterine lethal by 7 days post-coitis, but the heterozygous (Glut3+/-) littermate survives, exhibiting rapid post-natal weight gain, but no seizures or other behavioral aberrations. At 12 weeks of age, brain uptake of tail vein-injected 3 H-2-deoxy glucose in Glut3 +/- mice was not different from Glut3+/+ littermates, despite 50% less Glut3 protein expression in the brain. The brain uptake of injected 18F-2-fluoro-2-deoxy glucose was similarly not different from Glut3+/- littermates in the total amount, time course, or brain imaging in the Glut3+/- mice. Glut1 and Glut6 protein expressions evaluated by immunoblots were not affected by the diminished Glut3 expression in the Glut3+/- mice. We conclude that a 50% decrease in Glut3 is not limiting for the uptake of glucose into the mouse brain, since Glut3 haploinsufficiency does not impair brain glucose uptake or utilization.