Diffusion tensor imaging (DTI) has been proven to be a sophisticated and useful tool for the delineation of tumors. In the present study, we investigated the predictive role of DTI compared to other magnetic resonance imaging (MRI) techniques in combination with Ki-67 labeling index in defining tumor cell infiltration in the peritumoral regions of F98 glioma-bearing rats. A total of 29 tumor-bearing Fischer rats underwent T2-weighted imaging, contrast-enhanced T1-weighted imaging, and DTI of their brain using a 7.0-T MRI scanner. The fractional anisotropy (FA) ratios were correlated to the Ki-67 labeling index using the Spearman correlation analysis. A receiver operating characteristic curve (ROC) analysis was established to evaluate parameters with sensitivity and specificity in order to identify the threshold values for predicting tumor infiltration. Significant correlations were observed between the FA ratios and Ki-67 labeling index (r = - 0.865, p < 0.001). The ROC analysis demonstrated that the apparent diffusion coefficient (ADC) and FA ratios could predict 50% of the proliferating cells in the regions of interest (ROI), with a sensitivity of 88.1 and 81.3%, and a specificity of 86.2 and 90.2%, respectively (p < 0.001). Meanwhile, the two ratios could also predict 10% of the proliferating cells in the ROI, with a sensitivity of 82.5 and 94.9%, and a specificity of 100 and 88.9%, respectively (p < 0.001). The present study demonstrated that the FA ratios are closely correlated with the Ki-67 labeling index. Furthermore, both ADC and FA ratios, derived from DTI, were useful for quantitatively predicting the Ki-67 labeling of glioma cells.