Colorectal cancer (CRC) is the third most common cancer in the world with a higher prevalence in the developed countries, mainly caused by environmental and lifestyle factors such as diet, particularly red meat consumption. The metabolic impact of high red meat consumption on the epithelial part of the colon was investigated using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MSI), to specifically analyze the epithelial substructure. Ten colons from rats fed for 100 days high red or white meat diet were subjected to untargeted MSI analyses using two spatial resolutions (100 μm and 10 μm) to evaluate metabolite changes in the epithelial part and to visualize the distribution of metabolites of interest within the epithelium crypts. Our results suggest a specific effect of red meat diet on the colonic epithelium metabolism, as evidenced by an increase of purine catabolism products or depletion in glutathione pool, reinforcing the hypothesis of increased oxidative stress with red meat diet. This study also highlighted cholesterol sulfate as another up-regulated metabolite, interestingly localized at the top of the crypts. Altogether, this study demonstrates the feasibility and the added value of using MSI to decipher the effect of high red meat diet on the colonic epithelium.