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This study was designed to test whether metabolic characterization of intact, unaltered human rectal adenocarcinoma specimens is possible using the high-resolution magic angle spinning proton (1H) magnetic resonance spectroscopy technique.The study included 23 specimens from five patients referred for ultrasonographic staging of suspected rectal cancer. Multiple biopsies of macroscopically malignant rectal tumors and benign rectal mucosa were obtained from each patient for a total of 14 malignant and 9 benign samples. Unaltered tissue samples were spectroscopically analyzed. Metabolic profiles were established from the spectroscopy data and correlated with histopathologic findings.Metabolomic profiles represented by principle components of metabolites measured from spectra differentiated between malignant and benign samples and correlated with the volume percent of cancer (P = 0.0065 and P = 0.02, respectively) and benign epithelium (P = 0.0051 and P = 0.0255, respectively), and with volume percent of stroma, and inflammation.Magnetic resonance spectroscopy of rectal biopsies has the ability to metabolically characterize samples and differentiate between pathological features of interest. Future studies should determine its utility in in vivo applications for non-invasive pathologic evaluations of suspicious rectal lesions.