Glutamate excitotoxicity of cerebellar granule neurons (CGN) is often used as a model for studying the pathogenesis of acute, chronic neurodegenerative diseases and inflammation-induced neurological diseases. However, reports of culture methods for CGN and conditions for their stimulation vary considerably, making it difficult to compare reports from different laboratories. Here, we compare different methods and optimize the model as follows: Culture plates were coated overnight at room temperature with Poly-L-lysine at a concentration of over 50 mg/L. Cerebellum tissues were then shredded and digested with trypsin (0.025%) for 15 min at 37℃ with shaking in the presence of DNaseⅠ to eliminate DNA released from broken cells. In order to remove impurities and cell debris, samples were centrifuged respectively after tissue shredding, digestion and mechanical dispersion. Samples were then homogenized by pipetting and allowed to settle three times to increase the yield of CGN. Best results were achieved when settling time was extended to 15 min before collecting the supernatant cells. Conditional BME was used as the culture medium since our data showed that CGNs are more sensitive to glutamate in conditioned than in fresh BME, neurobasal medium or Locke's buffer. Cells cultured for 9 DIV (days in vitro) were treated with 100 μmol/L glutamate to generate moderate excitotoxicity. The model was validated by examining intracellular calcium dynamics and c-fos expression. Results show that this method is stable and reproducible and may be helpful for researchers using the excitotoxicity model to study neuropathological processes.