The primary aim of this study was to analyze human colon cancer and normal adjacent tissue using (1)H HR MAS MR spectroscopy and chemometric analyses, evaluating possible biomarkers for colon cancer. The secondary aim was to investigate metabolic profiles of tissue samples (n = 63, 31 patients) with microsatellite instability (MSI-H) compared to microsatellite stable (MSS) colon tissue. Our hypothesis was that this method may provide an alternative to MSI genotyping. Cancer samples were clearly separated from normal adjacent mucosa by 100% accuracy. Several metabolites such as lactate, taurine, glycine, myo-inositol, scyllo-inositol, phosphocholine (PC), glycerophosphocholine (GPC), creatine, and glucose were identified as potential biomarkers for cancer detection. Adenomas (n = 4) were also separated from cancer and normal samples mainly based on higher GPC and PC levels. Interestingly, metabolic differences in normal colon mucosa between MSI-H and MSS patients were observed. MSI status was validated with 80% accuracy with a sensitivity and specificity of 79% and 82%, respectively, including both cancer and normal samples The metabolic differences between MSI-H and MSS may be very interesting in the early detection of cancer development and of high clinical importance in the work of improving diagnosis and characterization of colon cancer.