Astrocytoma is the most common aggressive glioma and its early diagnosis remains difficult. Biomarkers are changes associated with the disease. Urine, which is not regulated by homeostatic mechanisms, accumulates changes and therefore is a better source for biomarker discovery. In this study, C6 cells were injected into Wistar rats brain as astrocytoma model. Urine samples were collected at day 2, day 6, day 10 and day 13 after injection, and the urinary proteomes were analyzed. On the 10th day, lesions appeared in magnetic resonance imaging. On the 13th day, clinical symptoms started. But differential urinary proteins were changed with the development of the astrocytoma, and can provide clues even on the 2nd and 6th day. Twenty seven differential proteins with human orthologs had been reported to associate with astrocytoma. A panel of differential urinary proteins may provide sensitive early biomarkers for the early diagnose of astrocytoma.