An experiment was designed to test the hypothesis that short-term oral administration of dietary vitamin D3 to beef cattle before slaughter would increase beef tenderness through greater calcium-activated calpain activity in postmortem aged skeletal muscle. Thirty continental crossbred steers were allotted randomly to three treatment groups housed in one pen. One group served as a control; two other groups were administered boluses with either 5 x 10(6) or 7.5 x 10(6) IU of vitamin D3 daily for 9 d. Cattle were slaughtered 1 d later. The longissimus lumborum was excised from each carcass 72 h postmortem and steaks removed at 3, 7, 14, and 21 d postmortem. The semimembranosus muscle (top round) was excised from each carcass 72 h postmortem and steaks removed at 7, 14, and 21 d postmortem. Blood plasma calcium concentration of cattle treated with 5 or 7.5 x 10(6) IU of vitamin D3 was higher (P < .05) than that of controls. Strip loin and top loin steaks from cattle fed supplemental doses of vitamin D3 had lower (P < .05) Warner-Bratzler (W-B) shear values at 14 d postmortem but were not significantly different from controls at 3, 7, or 21 d (strip loins) or 7 or 21 d (top rounds). No significant difference in strip loin steak tenderness was observed by sensory panel at 14 d postmortem (P < .17) between steaks from control and vitamin D3-treated steers. At 14 d postmortem, strip loin and top round steaks from cattle fed 5 x 10(6) IU of vitamin D3, but not from those given 7.5 x 10(6) IU, showed more proteolysis (P < .05) than did steaks from control cattle, based on Western blotting analysis. Therefore, the use of supplemental dietary vitamin D3 given daily for 9 d before slaughter did improve tenderness (lower W-B shear values) of 14-d postmortem aged beef. Increased proteolysis seems to be the mechanism of tenderization.