Objective: To study the expression of proliferating cell nuclear antigen (Ki-67) and glial fibrillary acidic protein (GFAP) and their pathological significance in patients with brain glioma. Methods: A total of 86 patients with brain malignant glioma in our hospital were selected and divided into a low-grade group and a high-grade group according to the tumor differentiation level of the patient. At the same time, 45 healthy and normal patients were selected as a control group, and their Ki-67 and GFAP expression were determined by immunochemical analysis. The Ki-67 and GFAP positive expression rates, Ki-67 and GFAP gray values, and the density of positive reactants were compared. Results: The Ki-67 positive expression rates, Ki-67 gray values and the density of positive reactants in the high grade and the low grade group were significantly higher than those in the control group (P<0.05). However, the GFAP positive expression rates, GFAP gray values and the density of positive reactants in the high grade group were significantly lower than those in the low grade and the control group (P<0.05). Conclusion: With the increase of Ki-67 and GFAP positive expression rate, the pathological grade of patients with brain glioma will increase. Ki-67 and GFAP can play a good guiding role in the identification of malignant brain gliomas, pathological grading and patient prognosis.