Impairment of cerebral glucose uptake/metabolism in individuals with Alzheimer's disease (AD) is believed to lead to downregulation of protein O-GlcNAcylation, which contributes to tau pathogenesis through tau hyperphosphorylation. Level of glucose transporter 3 (GLUT3), a neuronal specific glucose transporter, is decreased in AD brain, which may contribute to impaired brain glucose uptake/metabolism. However, what causes the reduction of GLUT3 in AD brain is not fully understood. Here, we report 1) that decrease of GLUT3 is associated with the reduction of protein O-GlcNAcylation in AD brain, 2) that GLUT3 level is negatively correlated with calpain I activation in human brain, 3) that calpain I proteolyzes GLUT3 at the N-terminus in vitro, and 4) that activation of calpain I is negatively correlated with protein O-GlcNAcylation in AD brain. Furthermore, we found that overexpression of GLUT3 enhances protein O-GlcNAcylation in N2a cells. Overexpression of calpain I suppresses protein O-GlcNAcylation in these cells. These findings suggest a novel mechanism by which calpain I overactivation leads to GLUT3 degradation and the consequent down-regulation of protein O-GlcNAcylation in AD brain.