Vitamin D3 was orally supplemented to determine the supplemental dose that improved beef tenderness in different cattle breed types. Feedlot steers (n = 142) were arranged in a 4 x 3 factorial arrangement consisting of four levels of supplemental vitamin D3 (0, 0.5, 1, and 5 million IU/steer daily) administered for eight consecutive days antemortem using three biological types (Bos indicus, Bos Taurus-Continental, and Bos Taurus-English). Warner-Bratzler shear force (WBSF) was measured at 3, 7, 10, 14, and 21 d postmortem, and trained sensory analysis was conducted at 7 d postmortem on LM, semimembranosus, gluteus medius, and supraspinatus steaks. Concentrations of vitamin D3 and the metabolites 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 were determined in the LM, liver, kidney, and plasma. Biological type of cattle did not interact (P > 0.10) with vitamin D3 supplementation for sensory or tenderness traits, suggesting that feeding vitamin D3 for 8 d before slaughter affected the different biological types of cattle similarly. Supplementing steers with 0.5, 1, or 5 million IU/(steer(d) decreased (P < 0.05) LM WBSF at 7, 10, 14, and 21 d postmortem compared with controls, and vitamin D3 treatments of 0.5, 1, and 5 million IU decreased (P < 0.05) semimembranosus WBSF at 3, 7, and 14 d postmortem. In general, vitamin D3-induced improvements in WBSF were most consistent and intense in LM steaks. Sensory panel tenderness was improved (P < 0.05) by all vitamin D3 treatments in LM steaks. Sensory traits ofjuiciness, flavor, connective tissue, and off-flavor were not (P > 0.05) affected by vitamin D3 treatments. All vitamin D3 treatments decreased micro-calpain activity and increased muscle Ca concentrations (P < 0.05). Vitamin D3 concentrations were increased (P < 0.05) by supplementation in all tissues tested (liver, kidney, LM, and plasma); however, cooking steaks to 71 degrees C decreased (P < 0.05) treatment residue effects. The vitamin D metabolite 1,25-dihydroxyvitamin D3 was increased (P < 0.05) only in plasma samples as a result of the vitamin D3 treatments. These results indicate that supplementation with vitamin D3 at 0.5 million IU/steer daily for eight consecutive days before slaughter improved tenderness in steaks from different subprimal cuts by affecting muscle Ca concentrations, micro-calpain activities, and muscle proteolysis, with only a small effect on tissue residues of vitamin D3.