Glucose transporters 1 (GLUT1) and 3 (GLUT3) belong to the solute carrier family 2 (SLC2, facilitated glucose transporter) and are the two most important glucose transporters (GLUTs) in brain tissue, and between them, GLUT3 is the primary one for neurons, which is responsible for glucose uptake. To obtain insights into the possible alterations of GLUT1 and GLUT3 in transmissible spongiform encephalopathies (TSEs), the protein levels of GLUT1 and GLUT3 in the brain tissues of agents 263K- and 139A-infected hamsters, as well as agents 139A- and ME7-infected mice, were evaluated. Western blots, immunofluorescent assay (IFA), and immunohistochemical (IHC) assays revealed that at the terminal stages of the infection, GLUT3 level in the brain tissues of scrapie-infected rodents was significantly downregulated, while GLUT1 level remained almost unchanged. The decline of GLUT3 level was closely related with prolonged incubation time. In line with these results in vivo, the GLUT3 level in a prion persistently infected cell line SMB-S15 was also lower than that of its normal cell line SMB-PS. Moreover, the level of hypoxia-inducible factor-1 alpha (HIF-1α), which positively regulated the expressions of GLUTs, was also markedly downregulated in the brains of several scrapie-infected animals. In vitro glucose uptake assays illustrated a markedly decreased 2-[N-(7-nitrobenze-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose uptake activity in SMB-S15 cells. Our data indicate that the reduction of GLUT3 is a common phenomenon in prion diseases, which occurs much earlier than the appearance of clinical symptoms. Defect in glucose uptake and metabolism of neurons, like in other neurodegenerative diseases, for example, Alzheimer's disease (AD), may be one of the essential processes in the pathogenesis of prion diseases.