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Schwann cells (SCs) isolation is one of the basic techniques for study of peripheral nervous system and peripheral neuropathy. A combined and effective method of isolating SCs from sciatic nerves of newborn mice with high yield and purity is still lacking. Sciatic nerves from neonatal mice aged 3–5 days serve as the source of SCs. Removal of adjacent connective tissue and epineurium, treatment with arabinoside hydrochloride and differential cell detachment technique were applied to eliminate fibroblast contamination and increase the purity of SCs. Combined use of collagenase/dispase and trypsin was chosen to increase the yield of SCs. Culture dishes precoated with poly-l-lysine and laminin, culture medium supplemented with heregulin β-1 and forskolin, and reasonable cell seeding density were implemented to increase the growth and proliferation of cultured SCs. Immunostaining of S100β and p75 neurotrophin receptor was used to identify the purity of SCs. Our method is able to obtain high-yield SCs with a purity of 90% within five days and a purity more than 99% within seven days from sciatic nerves of neonatal mice. Previous SCs isolation mostly focused on rats or adult mice and have a few limitations due to fibroblasts contamination, low yield and time-consuming. Our method permits SCs isolation from neonatal mice with a high yield and purity of primary SCs within 7 days. We described a fast, efficient and step-by-step method of isolating SCs from sciatic nerves of neonatal mice with high yield and purity. [Display omitted] • A new method of isolating Schwann cells (SCs) from newborn mice was described. • A high yield and purity (> 99%) of SCs isolation were obtained. • The whole procedure can be completely accomplished within 7 days. [ABSTRACT FROM AUTHOR]