Abstract: Objective: To establish a suitable primary culture method of rat cerebellar granule neurons. Methods: Rat cerebellar granule neurons were prepared from 5-7 day old Sprague-Dawley rat pups,the cerebella was freed of meninges,minced,trypsinized, then the cell suspension was preplated for 30 min for remove any glial cells,dissociated cells were seeded at plates which had been pre-coated with Poly-L-Lysine,arabinosylcytosine was added to the culture medium on day 3 after seeding for inhibition of non-neuronal cell division. Neurons were identified by neuron-specific enolase immunofluorescence technic. Results: The survival rate of the cells was (98 ±1.07)%; the neurons were affixed to the culture plate after 24 hours,neurite growth was apparently on day 3,integrated neural network was formed on day 6-8. Cerebellar granule neurons was about 90% by neuron-specific enolase identifying. Conclusions: Neuron purity was higher in the experiment; it is a perfect technique for primary culture of rat cerebellar granule neurons.