This study aimed to investigate the effects of 1,25-Dihydroxycholecalciferol glycoside (1,25(OH)D-G) supplementation on the performance, biochemical blood, bone health, intestinal histomorphometry, and gene expressions in broilers from breeders supplemented or not with 1,25(OH)D-G. A total of 1152 one-day-old, male Ross 308 AP chicks were distributed in a completely randomized design with a 2 × 3 factorial arrangement. One factor was the inclusion or not of 1,25(OH)D-G in the breeders' diets from 21 to 62 weeks of age. The other factor involved three levels (0, 50, and 100 mg/kg) of 1,25(OH)D-G inclusion in the broilers' diets from 1 to 21 days of age. The study totaled six treatments, with eight replicates and 24 birds per experimental unit. Breeders supplemented with of 1,25(OH)D-G resulted in heavier broiler chicks at hatch, better feed conversion, and higher concentrations of calbindin D28K, interleukin 10, and interleukin 1β at 21 days of age. Villus height and absorption area in the jejunum showed interactions between breeder supplementation and broiler diets. The inclusion of this additive in both diets was not sufficient to increase villus height and absorption area in broilers at 21 days of age. It is concluded that supplementation in breeders improves growth and immunity in broilers.