This study aimed to evaluate the effects of in ovo injection of soy isoflavones (ISF) on hatchability, body weight, antioxidant status and intestinal development of newly hatched broiler chicks. One hundred and eighty fertile eggs were divided as follows: the control group, 3 mg/egg ISF (low dose) and 6 mg/egg ISF (high dose) on the 18th day of incubation. The results demonstrated that in ovo inclusion of 6 mg of ISF significantly increased hatchability and hatch weight. Both doses of ISF inclusion elevated the serum glutathione peroxidase and slightly decreased malondialdehyde compared to the control group. The high dose of ISF brings higher villus height and a higher villus/crypt ratio in chicks. Moreover, the mRNA levels of tumour necrosis factor‐ α and interferon‐gamma in the spleen were significantly decreased. The ISF treatments showed an improvement in intestinal enzyme expression levels of sucrose isomaltase and mucin 2 as well as tight junction protein (TJ) mRNA expression of claudin‐1 at high doses of ISF (p < 0.05) when compared with the other groups. Furthermore, the mRNA level of IGF‐1 was increased in the high doses of ISF compared to the control. Overall, these findings indicate that in ovo administration of ISF on the 18th day of incubation enhances hatchability, antioxidant status and intestinal morphometrics in hatched chicks and modulates the expression of proinflammatory cytokines, TJs and insulin‐like growth factor. In addition, the sustainability of antioxidants and other positive effects of ISF may increase chick viability and growth performance.