Abstract: This experiment was performed to investigate the effects of combined treatment of high pressure and calcium choride(CaCl2) injection on post-rigor beef tenderness. Initially, samples from beef longissimus dorsi after 36 h post-slaughter were removed and the influences of CaCl2 concentration, high pressure level and high pressure processing time on beef tenderness were analyzed respectively. On the basis of the three single-factor experiments, the Box-Behnken response surface methodology was applied to optimize beef tenderization parameters. Meanwhile, the modifications of ultrastructure were observed with transmission electron microscope (TEM). The results indicated that the response surface model, with CaCl2 concentration, high pressure level and processing time being independent variables and shear force being responses, was highly fitted (R2=0.9742). Beef tenderness was significantly influenced by high pressure level, processing time, CaCl2 concentration, high pressure level × processing time (p<0.01), and high pressure level × CaCl2 concentration (p<0.05). The optimum tenderization parameters of combined high pressure and calcium chloride treatment were as follows: high pressure level was 241 MPa, CaCl2 concentration was 0.24 mol/L (with 5% injection of sample weight), processing time was 14 min, under which conditions, the shear force value of beef longissimus dorsi was declined by 52.98%, and intermyofibrillar space was enlarged, meanwhile the integrity of sarcomeres was destructed, and the tenderness was significantly improved.