The study investigated the effect of zingibain protease and sous vide cooking on tenderness and water-holding capacity of M. biceps femoris (BF) from 30 older Angus cows (6–7 years) fed concentrates for 0, 28, 42 or 56 days. BF were cooked for 1, 8, and 18 h at 65 °C and 75 °C, without any pre-treatment, after they have been injected with water only or after they have been injected with either 1 g/L or 2 g/L ginger powder solution (containing zingibain). Samples were tested for cooking loss, total water content, Warner-Bratzler shear force (WBSF), collagen content, and myofibrillar fragmentation index (MFI). Results revealed the significant interactions between concentrate feeding, ginger powder injection, cooking temperature, and time on quality traits. WBSF was reduced (P < 0.001) by increasing zingibain concentration at 65 °C but a gradual decrease was noted at 75 °C. Collagen solubility and MFI increased (P < 0.05) with increasing zingibain concentration. Injecting zingibain along with sous vide cooking demonstrated the weakening of myofibrillar and connective tissue proteins contributing to enhanced collagen solubility and tenderness in BF.