Tissue engineering of annulus fibrosus (AF), the essential load-bearing disc component, remains challenging due to the intrinsic heterogeneity of AF tissue. In order to provide a set of characterization data of AF tissue, which serve as the benchmark for constructing tissue engineered AF, we analyzed tissues and cells from various radial zones of AF, i.e., inner AF (iAF), middle AF (mAF), and outer AF (oAF), using a rabbit model. We found that a radial gradient in the cellular, biochemical, and biomechanical characteristics of rabbit AF existed. Specifically, the iAF cells (iAFCs) had the highest expression of collagen-II and aggrecan genes, while oAF cells (oAFCs) had the highest collagen-I gene expression. The contents of DNA, total collagen and collagen-I sequentially increased from iAF, mAF to oAF, while glycosaminoglycan (GAG) and collagen-II levels decreased. The cell traction forces of primary AFCs gradually decreased from iAFCs, mAFCs to oAFCs, being 336.6±155.3, 199.0±158.8, and 123.8±76.1 Pa, respectively. The storage moduli of iAF, mAF, and oAF were 0.032±0.002, 2.121±0.656, and 4.130±0.159 MPa, respectively. These measurements have established a set of reference data for functional evaluation of the efficacy of AF tissue engineering strategies using a convenient and cost-effective rabbit model, the findings of which may be further translated to human research.