Background and Objective: During the development of the nervous system, the impact of adverse environmental factors can cause serious consequences. Chemicals, toxic substances and infections are considered as common risk factors. In order to find the impact of environmental pollutants pesticides and infections on the nervous system, we attempt to investigate the effect of CPF (chlorpyrifos) and LPS (lipopolysaccharide) combined exposure on the release of GFAP/HMGB1 and activation in the primary cultured astrocyte of neonatal rat cerebral cortex. Methods: In this paper, trypsin digestion was used to isolate and purify astrocytes, and identify the cell based on immunofluorescence method; Astrocyte in experiment group was exposed to different concentrations of LPS and CPF. Astrocyte's activities were observed via CCK-8; Immunofluorescence and Western blot were used to measure the changes on the expression level of GFAP as well as HMGB1 in astrocyte. Results: Astrocyte's synaptic network was significantly increased at 24 h after being exposed to 25, 50 μMCPF combined with 1 μg/mL LPS; The expression level of glial fibrillary acidic protein (GFAP). High mobility group box 1(HMGB1) increased after astrocytes were exposed to 25, 50 μM CPF combined with 1 μg/mL LPS (p<0.05), The expression level of GFAP and HMGB1 decreased after being exposed to 100 μM CPF combined with 1 μg/mL LPS(p<0.05). 4) Conclusion: 1. CPF and LPS can not only cause astrocyte activation but also increase the expression of GFAP and HMGB1. 2. The effect of CPF combined with LPS is more obvious than CPF or LPS respectively on astrocytes, which indicates that they have a synergistic effect.