<p>Steak exhibits poor texture and tenderness due to high content of connective tissue and fascia, which reduce the acceptance of consumer. This study investigated the effects of papain-calcium chloride-citrus fiber complex tenderizer on steak quality and elucidated the tenderizing mechanism. Response surface optimization revealed that the optimal concentration ratio of the composite was papain (0.12%, w/w), CaCl₂ (0.13%, w/w), and citrus fiber (0.5%, w/w). Under the optimal composite treatment, the intermuscular gaps and sarcomere length of steak increased significantly. The myofibril fragmentation index and soluble protein content were enhanced by 2.3 times and 2.8 times respectively. The addition of the complex tenderizer promoted the conversion of free water to bound water in the steak, thereby enhancing its water holding capacity (from 66.99 ± 0.94 to 81.6 ± 0.33). The proportion of the α-helix elevated from 5.29 to 13.97% after tenderization treatment. Furthermore, the partial degradation of the myofibrillar protein occurred after tenderization treatment. The papain-calcium chloride-citrus fiber complex tenderizer can significantly improve the tenderness and water holding capacity of steak. Our results show that the compound tenderizer is a low-cost and convenient method for tenderizing steak, which provides a new perspective for improving the quality of steak.</p>