Objective To study the cell toxicity of thrombin in astrocytes in vitro and the protective effect of hirudo extract liquid (HEL) on the injured astrocytes. Methods Astrocytes were isolated from Wistar rats' cerebral cortex and cultured in vitro, and observed under a phase contrast microscope for growth status. Cell activity was measured with MTT assay. The survival of astrocytes was investigated after exposed to a selected concentration of thrombin ranging from 0.1 to 100 U/mL or to HEL ranging from 0.25 to 4 mg/μL by observing cell morphology under an inverted phase-contrast microscope and measuring the lactate dehydrogenase (LDH) activity (a marker of cell death) in cell supernatant. Expressions of HSP70 and TGFβ-1 protein in astrocytes were investigated by immunohistochemistry. Results (1) Thrombin (1-100 U/mL) had toxicity on astrocytes in vitro in a dose-dependent manner (F=118.65, P=0.000). (2) HEL (0.25-4 mg/μL) could significantly reduce the cell toxicity of 10 U/mL thrombin in astrocytes (F=156.08, P=0.000). With the increasing concentration of HEL, the protection of HEL was accordingly enhanced, and it even increased the expressions of HSP70and TGFβ-1. Conclusions HEL could accelerate the proliferation of astrocytes, enhance the expressions of HSP70 and TGFβ-1 protein, so as to significantly depress the cell toxicity of thrombin to astrocytes.