This study included two experiments, in order to investigate the effect of In ovo injecting of different levels of Acetic acid solution on hatchability, chicks characteristics and production performance of broiler chickens. The first experiment: In this experiment, 720 fertilized eggs were used from breeders of the broiler (Ross 308), distributed into six treatments, 120 egg per treatment by three replicates per treatment (40 eggs rep.-1), at the 18th day of incubation when the eggs are transferred from the setter to the hatcher, they were injected into the amniotic sac according to the experimental treatments, T1 (Negative control) hatching eggs without injection, T2 (Positive control) hatching eggs are injected 0.2 ml egg-1 distilled water only, and T3, T4, T5, T6 hatching eggs are injected 0.2 ml egg-1 of different concentrations of Acetic acid solutions achieves 1, 2, 3, 4 μl egg-1 respectively, after injection, the eggs were returned to the hatcher. The results showed a significant improvement (P≤0.05( in the Hatching Characteristics, body weight, body length, relative weight of the yolk sac, relative weight of the small intestine, length of the small intestine and humoral immunity in chicks of Acetic acid injection treatments compared with both control treatments (T1, T2). The second experiment: In this experiment, 255 broiler chicks Ross 308 were used, their took from the chicks in the first experiment of the best two treatments in the results that’s means the fifth and sixth treatments (T5, T6) as well as the treatment of control (T1) form randomly and an equal number of 75 chicks of each treatment, they were raised in standard conditions up to the age of 35 days. The results showed an improvement in the production performance of broiler chicks from the In ovo injection of Acetic acid treatments, which improved significantly (P≥0.05) each of the live weight, weight gain, feed intake, feed conversion ratio, mortality, production index, body fullness degree and the dressing percentage in broiler of both treatment which injected of acid (T2, T3) compared to birds of control treatment. In conclusion indicated that the process of coupling the In ovo injection technique with feeding Acetic acid solution to the chick embryos at 18th days of incubation, improve the hatchability, quality characteristics of the chicks and the production performance of broiler chickens.