ABSTRACT The purpose of this study was to determine if in ovo feeding with 25(OH)D3 could influence the incubation parameters of bone mineral composition, mineral density, and bone breaking strength in post-hatch broilers. Fertile eggs from Cobb® broiler breeders were either non-injected or injected with 0, 1.2, 2.4, or 3.6 μg of 25(OH)D3/100 μL olive oil at eight days of incubation. The data were subjected to variance analysis using the General Linear Model procedure (GLM) of SAS®. There was a contrast-1 effect (non-injected eggs vs. injected eggs) for tibial bone mineral density and egg mass loss, with higher bone mineral density in non-injected eggs (0.836 mmAl) compared with injected eggs (0.790 mmAl) and lower egg mass loss in non-injected eggs (11.25%) compared with injected eggs (12.10%). Tibial and femur bone mineral density responded quadratically, and injections of 0.47 and 0.68 μg of 25(OH)D3 increased tibia and femur bone mineral density, respectively. Egg mass loss responded quadratically, and 25(OH)D3 injections of 2.21 μg reduced egg mass loss (11.60%). In ovo feeding of 25(OH)D3 can compromise bone mineral density of post-hatch chicken.