Currently, the glucose measurements from the brain are mostly invasive or insufficient to provide high spatial-temporal information to interrogate areas of the brain. This study performed non-invasive glucose chemical exchange saturation transfer (glucoCEST) MRI without the need for a radioisotope and compared the results with the 2-deoxyglucose (2DG) autoradiography for measuring glucose levels in the rat model of mild traumatic brain injury (mTBI). Female 10-week-old SD rats (n=15) had mTBI using a 2m height/450g weight drop model. DTI and glucoCEST were acquired in vivo using a Bruker 7T scanner at baseline, 1, 10, 20, and 30 days-post-injury (DPI). Parameters for DTI: 3D spin echo EPI; TR/TE 700ms/37ms; b-value 800s/mm² with 17 encoding directions; voxel size 200 (μm, isotropic). Diffusion weighted images were corrected for B₀ susceptibility induced EPI distortion, eddy current distortions, and motion distortion with b-matrix reorientation using Tortoise.¹ CEST data were corrected for B0/B1 field inhomogeneity and WASSR water referencing.² Parameter for glucoCEST: 2D fast spin echo with (M_T) and without (M₀) magnetization transfer (MT) pulses (TR/TE 3.5s/11.5ms; in plane resolution: 200µm, thickness: 0.8mm; MT pulse: 2μT, 2s). The MT offset frequences (Δω) were set from -2kHz to +2kHz with 100Hz stepping to detect the proton metabolites of glucose (1.2ppm, 2.1ppm, 2.9ppm).³ Fractional anistropy (DTI-FA) and the asymmetry of magnetization transfer ratio (MTR_asym) were derived for mapping diffuse axonal injury (DAI) and glucose levels. After each scan, 3 rats were selected and processed for 2DG autoradiography (2DG-ARG). One-way ANOVA with repeated measures was performed by Prism v6.0. Except for those processed by the aforementioned software, all other imaging data were processed via in house Matlab scripts.Longitudinal T2W images were not different following mTBI, except for the subcutaneous edema at 1DPI showing hyperintensity (Fig 1A). DTI-FA maps clearly showed DAI in corpus callosum (outlined) from mTBI (Fig 1B). Compared to the baseline, the MTR_asym maps for glucoCEST revealed the glucose levels post injury that persisted up to 30DPI (Fig 1C). 2DG-ARG demonstrates a comparable trend of glucose levels indicating the hypometabolic areas following mTBI with nadir at 10DPI (Fig 1D). Compared to the baseline level, DTI-FA decreased 18% in corpus callosum at day 1 after trauma (Fig 2A). The MTR_asym progressively decreased down to 42% from day 1 to day 20 (Fig 2B). After reaching the lowest level at day 20, MTR_asym slightly returned to 63% of the baseline at day 30. 2DG-ARG substaintiates this trend showing comparable results that the lowest glucose uptake was seen at 10DPI and then normalized at 30DPI (Fig 2C).In mild TBI, energy substrate supply and consumption is crucial for the survival of traumatized brain. The widespread hypometabolism could affect the brain functions in learning and memory and result in progressive cerebral atrophy. The current glucoCEST results parallel with 2DG-autoradiography data showing glucose uptake largely decreased after TBI that persisted over 30 days. GlucoCEST produced higher image resolution and sensitivity MR images to monitor the changes of glucose metabolism in vivo, and be used to identify the potential window for effective treatments to increase the survival of stunned or injured parenchyma.