【Abstract】 Objective To investigate the best digestion time for isolation of annulus fibrosus cell and the effects of digestion time on the cell biological characteristics. Methods 20 New Zealand white rabbit were randomly divided into A, B, C, D and E group. After the rabbit was executed with air, The annulus fibrosus was taken from New Zealand white rabbits and digested with trypsase and collagenase Ⅱfor isolating annulus fibrosus cell. Type Ⅱ collagenase digestion time of A, B, C, D and E group were 1, 2, 3, 4 and 5 h, respectively. Then, the cells were cultivated in DMEM culture medium. By comparing the number of primary cell, cell survival rate, cell morphology and extracellular matrix expression, the suitable digestion time for isolating annulus fibrosus cell was determined. Results With the digestion time prolonging, the number of primary cell was increasing. The number of obtained cells was enough for C, D and E groups, but the cell adhesion for E group was undesirable. All groups were positive in toluidine blue staining and immunohistochemical staining of collagen Ⅰ andⅡ, and the staining for C and D group was deeper than other three groups. Conclusion During the process of annulus fibrosus cell isolation and cultivation by sequential digestion method, the best collagenase Ⅱ digestion time is 3 4h.