Degeneration of the intervertebral disk (IVD) is a major pathological process implicated in low back pain and is a prerequisite to disk herniation. Although mechanical stress is an important modulator of the degeneration, the underlying molecular mechanism remains unclear. The association of human IVD degeneration, assessed by magnetic resonance imaging, with annulus fibrosus cell apoptosis and anti-cytochrome c staining revealed that the activation of the mitochondria-dependent apoptosome was a major event in the degeneration process. Mouse models of IVD degeneration were used to investigate the role of the mechanical stress in this process. The application of mechanical overload (1.3 MPa) for 24 hours induced annulus fibrosus cell apoptosis and led to severe degeneration of the mouse disks. Immunostaining revealed cytochrome c release but not Fas-L generation. The role of the caspase-9-dependent mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload was investigated further with the use of cultured rabbit IVD cells in a stretch device. Mechanical overload (15% area change) induced apoptosis with increased caspase-9 activity and decreased mitochondrial membrane potential. Furthermore, Z-LEHD-FMK, a caspase-9 inhibitor, but not Z-IETD-FMK, a caspase-8 inhibitor, attenuated the overload-induced apoptosis. Our results from human samples, mouse models, and annulus fibrosus culture experiments demonstrate that the mechanical overload-induced IVD degeneration is mediated through the mitochondrial apoptotic pathway in IVD cells.