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Glioma is the most common primary intracranial tumor. Molecular neuropathology was introduced into the new 2016 WHO classification of brain tumor. Among the molecular biomarkers, Ki-67 antigen is the most important one, which reflects the proliferation rate and invasive ability of tumor cells. The amide proton transfer imaging, as a novel functional MR technique, can detect the free protein and polypeptide noninvasively, which might be a novel imaging method for predicting the WHO grading of glioma. In our study, the asymmetric magnetization transfer ratio (MTRasym) of high-grade gliomas (4.5%±2.3%) was significantly higher than of low-grade gliomas (2.9±1.1%), and the high-grade gliomas also showed higher expression of Ki-67 (38.9±21.0%) than did low-grade gliomas (4.3±2.8%). The MTRasym was positively correlated with Ki-67 values (r=0.25, P<0.001). The area under the receiver operating characteristic curve of MTRasym was 0.719. Furthermore, the diagnosis efficiency of MTRasym is better than that of the apparent diffusion coefficient (area under the receiver operating characteristic curve=0.682). This prospective study demonstrates that amide proton transfer may help in grading gliomas and has great potency in predicting tumor cell proliferation.