This study was conducted to investigate the role of vitamin D3 [25 (OH) D3]  on embryonic development, hatchability %, bone growth, and intestine histomorphology in broiler chickens. Forty hundred broiler breeder eggs (Ross-308; average egg weight: 63.2 g) were divided into four groups (100 eggs in 4 replicates of 25 eggs each) as follows: Control (eggs were not- injected); Vehicle: eggs were injected with 100 µl sterile distilled water (diluent); Vit-50 and Vit-100: eggs were injected with 50 and 100 µl of Vit. D3, respectively. The eggs were incubated in an automatic incubator at 99.8°F and 60% RH. On the 18th day of incubation, in ovo injection was applied to eggs with live embryos within the amnion sac. At hatch, hatchability %, chick weights, and plasma constituents were recorded. Afterward, at 35 days of age, performance and intestinal development were recorded. Results revealed that chick weight at hatch and hatchability (%) were not influenced by the treatments. Also, the results showed that plasma total protein, albumin, and globulin values of day-old chicks were significantly higher in chicks hatched from eggs that were injected with 100 µl of Vit. D3 compared with the other groups and this trend was also observed at 35 days of age. Furthermore, body weight, body weight gain, and feed conversion ratio were significantly improved in response to in ovo injection with vitamin D3. Moreover, plasma calcium and phosphorus were significantly increased in chickens that were injected with Vit. D3, either at one or 35 days old. Parathyroid hormone (PTH) concentration was significantly higher in day-old chicks that hatched from the control and vehicle groups, but the opposite trend was recorded at 35-day-old, where the Vit. D3 injected groups showed high levels. Weights and lengths of tibia, femur, and keel bones were significantly heavier and longer for broilers that hatched from eggs injected with Vit. D3 than the other treatments. Jejunum histomorphometry revealed significant changes in response to in ovo Vit. D3 injection, including villus length, width, villus length to crypt depth ratio, and villus surface area. It is concluded that in ovo injection on the 18th day of incubation had a significant improvement in productive performance, plasma protein, Ca, P, and PTH levels and villi characteristics were developed. However, there was no significant effect on chick weight or hatchability percentage