The objective of this experiment was to determine the effect of different doses of vitamin D3 (VITD) on beef feedlot performance, plasma and muscle Ca2+, tissue residues, and improvement of Warner-Bratzler shear force (WBS) and panel tenderness. A total of 167 steers were fed one of six levels of VITD. The VITD treatments (28 steers/treatment) were 0, 0.5 x 10(6), 1 x 10(6), 2.5 x 10(6), 5 x 10(6), and 7.5 x 10(6) IU/steer daily of VITD fed nine consecutive days before slaughter. Feedlot performance and plasma Ca2+ were measured during the last 21 days on feed. Warner-Bratzler shear force was measured on strip loin and top round steaks at 7, 10, 14, and 21 d postmortem. The VITD treatments of 5 and 7.5 x 10(6) IU/steer daily decreased (P < 0.05) ADG, and VITD supplementation of 2.5, 5, and 7.5 x 10(6) IU/steer daily decreased average dry matter feed intake (P < 0.05) at the end of the feeding trial. Plasma Ca2+ increased linearly with VITD treatment (P < 0.01). Calpastatin and calpain activity were not influenced by treatment (P > 0.05), but muscle Ca2+ was increased (P < 0.05) by VITD treatments of 1, 2.5, 5, and 7.5 10(6) IU/steer daily. Feeding VITD did not influence (P > 0.05) carcass quality or yield traits. Supplementing VITD at levels of 1, 2.5, 5, and 7.5 10(6) IU/steer daily increased (P < 0.05) VITD concentrations in strip loin and liver samples. Cooking liver decreased VITD concentrations 10 to 28%. Vitamin D3 treatments of 0.5 and 7.5 x 10(6) IU/d reduced strip loin steak WBS at d 7 (P < 0.05), but VITD treatments did not decrease strip loin steak WBS at any other time postmortem. The VITD treatments of 0.5, 1, and 5 x 10(6) IU/steer daily decreased top round steak WBS at 7 d, and all VITD treatments decreased 10-d top round steak WBS (P < 0.05). Supplementing steers with 0.5 x 10(6) IU/steer daily of VITD also decreased (P < 0.05) top round steak WBS at 21 d postmortem compared with controls. Sensory tenderness at 7 d postmortem was increased (P < 0.05) by all VITD treatments in top round steaks, yet strip loin tenderness scores were not affected (P > 0.05) by VITD treatment. Treatment with VITD quadratically decreased (P < 0.05) round WBS. Thus, VITD treatment will effectively improve tenderness when cattle tend to be tough and have no impact on cattle that produce tender beef. Feeding steers 0.5 x 10(6) IU of VITD daily for 9 d improved tenderness in two muscles without negatively affecting feedlot performance or tissue residues.