Improving tenderness of beef from cows is desirable because it could increase marketability of these carcasses and, thereby, increase profits to producers. In this study, two dietary treatments, supplemental 25-hydroxyvitamin D3 (25-OH D3) and manipulations of dietary calcium, were examined as methods of increasing plasma and muscle calcium concentrations as a means for improving tenderness of beef. Our results indicate that the environment created in the muscle by supplementing 25-OH D3, increasing dietary calcium, or both is conducive to increased activity of calpain, a calcium-dependent enzyme responsible for postmortem tenderization of beef. Increased calpain activity could lead to improved beef tenderness.