Intervertebral disc degeneration is associated with back pain and disc herniation. This study established a modified protocol for intervertebral disc (IVD) decellularization and prepared its extracellular matrix (ECM). By culturing mesenchymal stem cells (MSCs)(3, 7, 14 and 21 days) and human degenerative IVD cells (7 days) in the ECM, implanting it subcutaneously in rabbit and injecting ECM microparticles into degenerative disc, the biological safety and efficacy of decellularized IVD was evaluated both in vitro and in vivo. Here, we demonstrated that cellular components can be removed completely after decellularization and maximally retain the structure and biomechanics of native IVD. We revealed that allogeneic ECM did not evoke any apparent inflammatory reaction in vivo and no cytotoxicity was found in vitro. Moreover, IVD ECM can induce differentiation of MSCs into IVD-like cells in vitro. Furthermore, allogeneic ECM microparticles are effective on the treatment of rabbit disc degeneration in vivo. In conclusion, our study developed an optimized method for IVD decellularization and we proved decellularized IVD is safe and effective for the treatment of degenerated disc diseases.