The objective of this study was to investigate the effect of atractyloside, a promoter of the mitochondrial membrane permeability transition pore (MPTP), on mitochondrial membrane permeability, cristae structure, cytochrome c release and tenderness of postmortem beef and to explore the effect of injury on meat tenderness by releasing cytochrome c and activating caspases. The postmortem longissimus dorsi muscle of adult crossbred cattle was treated or not treated (control) with atractyloside, stored at 4 ℃ and evaluated for mitochondrial ultrastructure, membrane potential, the amount of cytochrome c released to the cytoplasm, cell apoptotic rate, caspase-3 and myofibrillary fragmentation index (MFI) after 0, 12, 24, 72, and 120 h. With postmortem aging time, the number of mitochondrial cristae decreased significantly, and swelling and rupture were observed. The mitochondrial membrane potential decreased significantly, and cytochrome c was released gradually. Starting from 72 h postmortem, the activity of caspase-3 and the apoptosis rate of myocytes increased, and so did MFI. Compared with the control group, atractyloside treatment induced mitochondrial damage by destroying mitochondrial cristae structure, reducing the membrane potential and causing cytochrome c release. In addition, the apoptosis of muscle cells and the fragmentation of myofibrils were accelerated by atractyloside. The opening of the MPTP significantly affected mitochondrial injury in beef after slaughter. Apart from increasing cell membrane permeability, atractyloside also significantly altered the structure of mitochondrial cristae. The internal and external mitochondrial membranes could synergistically regulate the release of cytochrome c, which in turn could affect postmortem tenderization of beef.