The objective of this trial was to determine whether a single bolus of 25-hydroxyvitamin D₃ (25-OH D₃), vitamin E, or a combination of the 2 would improve the tenderness of steaks from the LM of beef heifers. Forty-eight Angus crossbred heifers were allotted randomly to 8 pens. Six heifers were in each pen, and there were 2 pens per treatment. The 4 treatments included control (no 25-OH D₃ or vitamin E); 25-OH D₃ (500 mg of 25-OH D₃ administered as a one-time oral bolus 7 d before slaughter); vitamin E (1,000 IU of vitamin E administered daily as a top-dress for 104 d before slaughter); or combination (500 mg of 25-OH D₃administered as a one-time oral bolus 7 d before slaughter and 1,000 IU of vitamin E administered daily as a top-dress for 104 d before slaughter). Blood samples were obtained on the day that heifers were allotted to treatments, on the day 25-OH D₃ was administered, and on the day before slaughter. Plasma calcium concentration was increased when 25-OH D₃ was administered with or without vitamin E (P < 0.007). In LM, calcium concentration tended to increase (P = 0.10) when 25-OH D₃ was administered alone but not when 25-OH D₃ was administered with vitamin E. Concentrations of 25-OH D₃ and 1,25-dihydroxyvitamin D₃ in plasma were increased when 25-OH D₃ was administered with or without vitamin E (P < 0.001). Steaks from heifers treated with 25-OH D₃or vitamin E, but not both, tended to have lower Warner-Bratzler shear force than steaks in the control group at 14 d postmortem (P = 0.08). Postmortem protein degradation as measured by Western blot of the 30-kDa degradation product of troponin-T was increased with all treatments after 3 d postmortem (P ≤ 0.07), but not at 7 or 14 d postmortem. Unexpectedly, the use of 500 mg of 25-OH D₃ fed as an oral bolus 7 d before slaughter or 1,000 IU of vitamin E administered daily for 104 d before slaughter alone, but not in combination, effectively decreased Warner-Bratzler shear force.