Microglia, implicating in such neuro-pathologies as brain inflammation, neurodegeneration, glioma, and neurogenesis, play an important role in central nervous system. Advanced research on microglia is crucial in exploring the neuro-pathology and neuro-physiology of these diseases, so how to culture large numbers of microglia in vitro becomes the base of a research. The wildly used method, at present, obtaining microglia from murine cannot fulfill the requirement of research, costing too much time and needing too many rats. We intend to introduce an optimized method that can harvest large quantities of microglia with high purity. Neonatal 2-3 days old Wistar rats were sacrificed and the cerebral cortices were trypsinized. We primarily cultured mixed cortical cells for 8-10 days. The microglia were harvested from the liquid supernatant; the left cells in the mixed cortical glial culture were passaged at a 1:2 density. After another 8-10 days of culture, microglia were collected again. And then, we passaged the left cells again for acquiring microglia from the third collection. We did not add additional mitogens in the experiment. At last, on average, 7.0 × 10(6) microglia were collected from one neonatal rat. By this modified method, much more microglia can be effectively and easily harvested comparing with the usual protocol before. We compared the characteristics of microglia harvested from these three passages, such as morphology, phenotype, purity, and abilities on proliferation, secretion, and phagocytosis. The cells presented typical microglia morphology, having phenotype markers of CD11b/c and CD45. The microglia from these three passages retained similar phagocytosis and secretion functions. Expanded population of microglia for investigation can be provided by this easy method in a short time with little cost and few rats.