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Objective To establish a simple , practical , highly-effective and stable method for the isolation and cultivation of rat hair follicle cells. Methods Under sterile condition, single hair follicle was taken out after the skin around the barbel of SD neonatal rats was sheared off. And then the hair follicles were digested with two-step method with Type Ⅰcollagenase and trypsin. The obtained cell suspension was planted into the culture plate which was covered with extracellular matrix, and then was cultivated with K-SFM culture medium containing fetal bovine serum with volume fraction of 1%. On the next day, K-SFM culture medium was replaced with serum-free culture medium. The remaining tissues were cut into pieces and spread out in the culture flask, and then were cultivated with HG-DMEM culture medium containing serum. Two kinds of cells were harvested and then were identified by immunofluorescence. The hair follicle epithelial cells were tested by flow cytometer. Results The hair follicle epithelial cells obtained through the above methods showed rapid adherence, and were round or polygon-like , with typical cobblestone-like morphology. The long spindle-shaped cells were seen around the tissues cultivated, having many protrusions on the surface of the cells, and they were interconnected into reticular structure. The expression of cytokeratin 15, cytokeratin 19 and β1 integrin in epithelial cells were positive. Most of the epithelial cells were in the G1 phase, accounting for 75.6%. The expression of laminin ( LN), fibronectin ( FN) and vimentin in the connective tissue sheath cells were also positive. Conclusion The cells harvested by modified two-step enzyme digestion method have confirmed as hair follicle cells and fibroblasts, and the obtained cells are of rapid adherence, good homogeneity, and active proliferation.