Objective To observe the proliferation, migration and differentiation of highly enriched oligodendrocyte precursor cells (OPCs) in vitro. Methods OPCs were dissociated and purified by shaking method from cultured glia mixture, which was derived from postnatal 1 day SD rats. BrdU incorporation assay was applied to analyze the proliferation ability of OPCs. Transwell and reaggragration assay were employed to determine the migration ability of OPCs. In differentiation model, the maturation ability of OPCs was examined by immunostaining and Western blotting analysis of myelin basic protein (MBP). Results The stratification of glia mixture occurred 10 days after culture. The microglia was removed by first session of shaking, and there were multiple aggregated OPCs scattered on the surface of astrocyte layer. The separated OPCs were positive for NG2 and A2B5. Ara-C greatly inhibited the proliferation of OPCs as observed by BrdU incorporation assay. PDGF obviously promoted migration of OPCs in both Transwell assay and reaggregation models. The ratio of MBP positive cells (mature oligodendrocytes) was increased in the differentiation medium. Besides, Western blotting analysis showed that MBP in differentiation medium was greatly elevated compared to that in proliferation medium (P<0.01). Conclusion The in vitro cultured and purified OPCs can maintain their fundamental characteristics of proliferation, migration and differentiation.