To study the correlation between apparent diffusion coefficient (ADC) and Ki-67, a marker of tumor activity, in the diagnosis of gliomas. Conventional magnetic resonance imaging (MRI), enhanced scanning, and diffusion-weighted imaging were performed in 76 patients with pathologically confirmed gliomas. The ADC values were measured at tumor parenchyma and the corresponding contralateral normal brain. The relatively ADC (rADC) values of the tumor parenchyma were calculated. The correlation of the ADC values with tumor grades was analyzed. The expression of Ki-67 was detected by immunohistochemical staining. The correlation between ADC value and Ki-67 in the diagnosis of gliomas was analyzed. The ADC values and rADC values of high-grade gliomas were significantly lower than those of low-grade gliomas. The ADC values of tumor parenchyma were inversely associated with the degree of malignancy of the gliomas (r=-0.898, r=-0.868; P<0.01). The expression of Ki-67 was significantly higher in high-grade gliomas than that in low-grade gliomas. The Ki-67 labeling index in grade 3 and 4 gliomas were (29.48 ± 19.78)% and (31.21 ± 17.50)%, respectively. Both of them were significantly higher than Ki-67 labeling index in low-grade (grade 1 and 2) gliomas [(2.33 ± 2.20)%] (P<0.01). Nevertheless, the Ki-67 labeling index showed no significant difference between grade 3 and 4 gliomas (P>0.05). The expression of Ki-67 was negatively correlated with the ADC values and rADC values in tumor parenchyma (r=-0.627, r=-0.607; P<0.01). The ADC and rADC values of tumor parenchyma can indirectly reflect the proliferation and malignancy of gliomas and therefore can be useful for the grading of glioma.