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The present study was conducted to investigate the effects of in ovo injection of ascorbic acid on antioxidant capacity and immune-related gene expression in the newly hatched local Chinese yellow broiler chicks. Fertile Chinese yellow broiler eggs (n = 90) were assigned to three equal groups. The first group was a non-injected control group. The second group was another control group where the eggs were injected with saline in the air sac after 18 days of incubation. The third group was injected with 3 mg/egg AA in the air sac after 18 days of incubation. In ovo injection of 3 mg/egg AA significantly (P < 0.0001) increased plasma glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and significantly reduced plasma malondialdehyde (MDA) at 1 d old. Moreover, in ovo injection of 3 mg/egg AA significantly increased mRNA expression of glutathione peroxidase (GSH-PX) and superoxide Dismutase (SOD) in the chick's spleen.Additionally, the mRNA level of interleukin 1 β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor α (TNF-α) in the spleen were significantly decreased (P < 0.0001), which indicates an improvement in chick's immunity. In conclusion, our data suggest that in ovo injection of AA at 3 mg/egg enhance antioxidant defense system and immune system for newly hatched chicks.