This study aimed to assess the effects of in ovo injection with vitamin D₃ on Norfa chick's performance. All fertile eggs were kept at incubation room temperature for 12 hours and then incubated in a forced draft incubator at 37.8°C with 55 - 60% relative humidity. Eggs were turned every two hours from the 2^nd to 18^th day of incubation. In the 1^st group, 40 eggs were not injected and considered as negative control, while in the 2^nd group, 40 eggs were injected with distilled water (positive control). In the 3^rd and 4^th groups, 160 and 180 eggs were injected with 50 and 100µL vitamin D₃ (VD₃), respectively. Eggs were injected on the 18^th day of incubation to deposit test material in the air sac through the wide end of the egg then the hole was closed by wax. During the last 3 days of incubation, all eggs were transferred to a separate hatcher at 36.5°C and 75 - 80% relative humidity. At hatch, the chicks were wing-banded, weighed and kept in the brooding house. The results showed that the chicks produced from eggs injected with 100 µL VD₃ exhibited significantly greater live body weight and body weight gain compared to other groups at different ages. Moreover, the feed conversion ratio and embryonic mortality rate were significantly affected during experimental period. The embryonic mortality rate was significantly increased in all treated-groups compared to negative control group. Immune response at 7 days and 28 days for Norfa chicks injected with VD₃ significantly improved compared to control groups. The highest value for femur and sternum bone length was recorded in chicks injected with 100µL VD₃, while tibia bone length was significantly decreased. Blood AST and ALT levels for chicks injected with 100µL VD₃ significantly increased compared to negative control group. In ovo injected VD₃ of 50 and 100 µL increased total serum calcium compared with the control groups. These results indicated that the in ovo injection of different levels of vitamin D₃ had a significant effect on white blood cells, lymph count, neutrophil, red blood cells and platelets. However, there was no significant effect of the in ovo injection on the levels of hemoglobin and hematocrit. In conclusion, early in ovo injection of different levels of vitamin D₃ might be considered a feasible technique for increasing Norfa chicks performance, hatchability, blood parameters, immunity and decreasing mortality.