Research on changes to glutamate signaling in the white matter of demyelinating diseases may provide important biophysical information for diagnostic and prognostic assessment. We attempted to evaluate glutamate signals in a cuprizone-induced rat model of demyelination by GluCEST imaging. GluCEST imaging provides in vivo image contrast of changing glutamate concentrations during demyelination and subsequent remyelination. We also performed histological validation to analyze the state of myelinated axons. GluCEST imaging could be a useful tool to evaluate brain metabolism in demyelination and remyelination models and provide quantitative results that are highly representative of changes to glutamate levels in vivo.