The objective of this trial was to determine how 25-hydroxyvitamin D3(25-OH D3) supplementation, altering supplemental dietary calcium, or their combination influence postmortem biochemical and tenderness changes in muscles from the round of mature cows. Twenty-seven Angus cows (3 to 7 yr old) were allotted randomly to 9 pens with 3 cows per pen. Treatments were arranged in a 3 × 3 factorial design with 3 dosages of 25-OH D3 (0, 250, or 500 mg of 25-OH D3 administered as a 1-time oral bolus 7 d before slaughter) and 3 percentages of supplemental limestone (0.5, 0.75, and 1.0%) replenished in the diet for 3 d before slaughter and after a 2-wk limestone withdrawal. Plasma samples were obtained during the feeding period. Upon slaughter, adductor, gracilus, pectineus, sartorius, semimembranosus, vastus intermedius, and vastus lateralis muscles were obtained and aged for 1, 3, or 7 d. Calcium concentrations were increased in plasma when 250 or 500 mg of 25-OH D3 were administered (P ≤ 0.05). Calcium concentrations in muscle increased (P ≤ 0.001) when 500 mg of 25-OH D3 were administered. Concentrations of 25-OH D3 in meat and in plasma and 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] in plasma were increased when 25-OH D3 was administered (P≤ 0.05). The percentage of limestone replenished in the diet had no effect on 25-OH D3 or 1,25-(OH)2 D3 in meat or in plasma. Calpastatin activity was affected by treatments only in the gracilus and vastus intermedius muscles (P ≤ 0.05). Among all muscles and aging periods, calpastatin activity and intensity of troponin-T degradation product were related inversely. Results indicate that supplemental 25-OH D3 has some influence on muscle characteristics known to improve tenderness, but improved tenderness was not observed.