Objective: <i>Bacillus subtilis</i>, a kind of probiotic with broad-spectrum antibacterial function, was commonly used in livestock and poultry production. Recent research suggested that <i>Bacillus subtilis</i> may have antioxidant properties and improve immune response. This study aimed to verify the probiotic function of <i>Bacillus subtilis</i> in the production of broiler chickens.Methods: A total of 324 (1-day-old) Arbor Acres broilers were selected and randomly divided into three groups: basal diet group (Ctr Group), basal diet + antibiotic growth promoter group (Ctr + AGP) and basal diet + 0.5% <i>Bacillus subtilis</i> preparation group (Ctr + Bac). The experiment lasted for 42 days. Muscle, serum and liver samples were collected at 42 days for determination.Results: The results showed that <i>Bacillus subtilis</i> could decrease malondialdehyde content in the serum and liver (p<0.05) and increase superoxide dismutase 1 mRNA expression (p<0.01) and total superoxide dismutase (p<0.05) in the liver. In addition, compared with AGP supplementation, <i>Bacillus subtilis</i> supplementation increased interleukin-10 (IL-10) and decreased tumor necrosis factor-α and IL-1β level in the serum (p<0.05). At 45 minutes after slaughter Ctr + Bac presented a higher a* value of breast muscle than Ctr Group (p<0.05), while significant change in leg muscle was not identified. Moreover, there was no difference in weight, shear force, cooking loss and drip loss of breast and leg muscle between treatments.Conclusion: Our results demonstrate that <i>Bacillus subtilis</i> in diet can enhance antioxidant capacity and optimize immune response of broilers.