The present study was focused on evaluating the effects of Bacillus methylotrophicus SY200 in broiler production. A total of 120 healthy 7-day-old broiler chicks were randomly assigned to four dietary treatments, which included basal diet supplemented with 0%, 0.10%, 0.25%, or 0.50% (w/w) B. methylotrophicus SY200 preparation (1.0 × 109 cfu/g), regarded as negative control group (NC), low-dose group (BML), medium-dose group (BMM), and high-dose group (BMH), respectively. Each treatment was fed the corresponding experimental diet for 35 days. Results showed that dietary supplementation of B. methylotrophicus SY200 could improve broiler weight gain, especially the finisher phase. Further studies suggested that a certain amount of B. methylotrophicus SY200 enhanced the broiler antioxidant status and improved the morphological development of jejunum. Besides, dietary supplementation of B. methylotrophicus SY200 especially in 0.50% levels significantly increased the relative weight of immune organs and Newcastle disease virus antibody titer, similarly, increased mRNA expression levels of claudin-1, claudin-3, zonula occluden-1, and zonula occluden-2 were observed in the jejunum of BMM group. Moreover, B. methylotrophicus SY200 also showed beneficial effects in improving broilers microbiota homeostasis by increasing the number of beneficial bacteria. Conclusively, B. methylotrophicus SY200 could effectively improve the antioxidant status, modulate the intestinal structure, enhance the intestinal mucosal barrier function, and regulate the immune function of broilers, which finally improves the performance of the chicken in the finisher period.