The aim of this study was to investigate the inhibitory effects of recombinant adenovirus vector carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) against degeneration of rabbit intervertebral disc. Thirty Japanese white rabbits of 4 months old were randomly divided into 5 groups. Mild or moderate rabbit lumbar disc degeneration model was constructed with the controllable axial loading device by imposing 98N pressure at the discs for 2 weeks. Various doses of virus were injected into the degenerated discs as follows: 20μL of normal saline in group 1; 20μL of RAd66 (an empty adenovirus vector, 1.0×10OPU/mL) in group 2; and 20, 10, and 5μL of RAdTIMP-3 (1.0×10OPU/mL) in groups 3, 4, and 5, respectively. Two weeks after the injection, the discs were collected for investigations, including assessment of degeneration degrees according to the Thompson’s grading system, reverse-transcription polymerase chain reaction (RT-PCR) assay for TIMP-3 gene, Safranin O-Fast green staining, and immunohistochemical staining for TIMP-3 and type II collagen. According to Thompson’s criteria, the degeneration of groups 3, 4, and 5, especially group 3, was alleviated as compared with groups 1 and 2. RT-PCR revealed that the expression of TIMP-3 in groups 3, 4, and 5, especially in group 3, was significantly enhanced as compared with group 1 (<0.01). Both Safranin O-Fast green staining and type II collagen staining demonstrated better reserved integrity of disc matrix in groups 3, 4, and 5 than in groups 1 and 2. TIMP-3 staining exhibited an obvious increase of positive-staining rate in groups 3, 4, and 5 as compared with group 1. The positive-staining rate in group 3 (79.42%±1.35%) was about 3times that of group 1 (25.47%±5.46%, <0.01). RAdTIMP-3 can effectively protect the matrix of rabbit intervertebral disc against overloading-induced degeneration in a dose-dependent manner, resulting in the alleviation of disc degeneration.