The aim of this study was to determine the effect of dietary vitamin D 3 supplementation on changes in color and heme pigment (myoglobin and metmyoglobin) in 4 beef muscles. The muscles came from 48 crossbred bulls that were subjected to supplementation with vitamin D 3 at 4 different doses: 0.0 (control group), 3.5, 7.0, and 10.0 × 10 6 IU. The 3.5 × 10 6 IU dose was administered daily for 6 days, before the morning feeding. Four days before slaughter, the supplementation was withdrawn. The dose of 7.0 and 10.0 × 10 6 IU was administered for 3 days and withdrawn 7 days before slaughter. The data show that supplementation with vitamin D 3 had a negative effect on beef meat color (P < 0.05). All analyzed muscles with vitamin D 3 had lightness (L*) and yellowness (b*) values higher than those of the control group (P < 0.05). Furthermore, redness (a*) decreased and metmyoglobin increased as a function of vitamin D 3 dose. These negative effects on meat color occurred with 7.0 and 10.0 × 10 6 IU doses. The muscle microstructure was imaged and it showed the effects of higher Ca 2+ concentrations on the deterioration of meat color.