Objective To investigate effects of different de-cellularization methods on biomechanical properties and histological structure of annulus fibrosus in pigtails and provide experimental evidence for the construction of tissue engineering annulus fibrosus. Methods Sixty Fresh annulus fibrosus were dissected from caudal disks of pigs and randomly assigned to 4 groups with 15 in each group. Triton X-100 group(Group A): annulus fibrosus were treated with hypotonic Tris-HCl buffer for 48 hours and de-cellularized with Triton X-100, DNase Ⅰ and RNase A. SDS group (Group B): annulus fibrosus were subjected to 3 cycles of freeze-thaw and subsequently de-cellularized with SDS, DNaseⅠ and RNase A. Trypsin group (Group C): annulus fibrosus were de-cellularized with Tris buffer containing trypsin, DNase Ⅰ and RNase A. Control group: fresh annulus fibrosus underwent no treatment. After the de-cellularization process was completed, hematoxylin-eosin (HE) staining was carried out to examine the efficacy on cell removal, and the ultrastructure of annulus fibrosus were observed by scanning electron microscopy. The collagen content, glycosaminoglycan (GAG) content and biomechanical parameters in each group were also detected. Results HE staining and scanning electron microscopy showed that no residual cells were found in Group A, B and C. The structure of annulus fibrosus in Group A was not disturbed, while that in Group B and C was damaged severely and slightly, respectively. There was no statistical difference in collagen content among Group A, B and C, as compared to the control group (P>0.05). But the GAG content was significantly more lower in Group A, B and C than in the control group (P<0.05). There was no statistical difference in ultimate load, ultimate stress, toughness, elastic modulus and mechanical work to fracture between Group A, C and control group (P>0.05), while these parameters of Group B were lower than those in the control group (P<0.05). Conclusions The Triton X-100-treated annulus fibrosus retained the major extracellular matrix composition after cell removal and preserved the major structure and mechanical strength, which is preferable for the construction of tissue engineering annulus fibrosus.