〔Abstract〕 Objective To explore and optimize thein vitroprimary culture method of astrocytes in neonatal mouse cerebral cortex, which provides a better solution for thein vitroculture of astrocytes. Methods In order to optimize thein vitroculture method of mouse cerebral cortex astrocytes, 3-day-old C57BL/6J mouse cerebral cortex tissues were taken, meninges and blood vessels were removed, digested by pancreatic enzymes and centrifuged, and high-glucose dulbecco′s modified eagle medium(DMEM) was added to form cell suspension, which was purified by differential adhesion method, cross hand method and constant temperature shaking method. The cells were inoculated in poly-D-lysine-coated culture bottles with different culture densities, and the purity of astrocytes was determined by morphological observation and immunofluorescence staining. Results The cells were inoculated at a density of 5×106cells per bottle with good effect and high activity. The purity of astrocytes reached 99% by using high sugar DMEM medium combined with differential adhesion method, cross hand method and constant temperature shaking method. Conclusion The primary culture method of astrocytes in mouse cerebral cortex is successfully established and optimized.