The objective was to evaluate the effect of dietary 25-hydroxycholecalciferol (25(OH)D 3 ) or vitamin D 3 (VitD 3 ) and different total calcium (Ca) levels on the performance, carcass characteristics, blood, enzymatic, and bone biochemistry of broilers reared under heat stress between 1 and 42 days of age. A total of 504 male, Cobb 500, broiler chickens were distributed in a completely randomized design in a 2 × 4 factorial arrangement (VitD 3 or 25(OH)D 3 × four Ca levels (100, 90, 80 and 70% of the recommendations of Rostagno et al. ( 2011)), eight treatments, seven replicates and nine broilers per cage. Feed intake and feed conversion ratio did not (p>0.05) vary when levels of Ca were reduced and vitamin D 3 sources were supplemented in the diets from 1 to 21 days for broilers chickens. 25 (OH)D 3 increased weight gain results (p<0.05). From 1 to 42 d, no differences (p>0.05) were observed on performance, carcass yields and meat quality, bone deposition of Ca and P, and alkaline phosphatase concentration. Higher serum (p<0.05) concentrations of Ca and P were found in broilers fed with 25(OH)D 3 . The replacement of VitD 3 with 25(OH)D 3 and the Ca reduction of 30% in diets did not negatively affect performance, carcass characteristics, and Ca and P deposition in the tibia of broilers at 42 days of age, under heat stress.