Aim: This study was conducted to investigate the effects of Clostridium butyricum in isolation or in combination with 1, 25-dihydroxyvitamin D 3 in early-stage broilers. Methods and Results: A total of 360 half male and half female Cobb broilers (1 day old) were randomly divided into four groups: Con (basal diet), Anti (basal diet+75 mg/ kg chlortetracycline), Cb (basal diet+10 9 CFU per kg C. butyricum) and CD (basal diet+10 9 CFU per kg C. butyricum+25 μg/kg 1,25(OH) 2 D 3 ). The results were as follows: (1) Compared with Con, CD significantly increased ADG (p < 0.05). ( 2) Contrast with Con and Anti, Cb and CD significantly increased glutathione peroxidase and SOD in the serum and liver, and decreased malondialdehyde content in serum (p < 0.05). (3) In addition, the content of immunoglobulin (IgA, IgY and IgM) in Cb and CD birds was higher than that in Con birds (p < 0.05); the Cb supplementation decreased (p < 0.05) the contents of IL-8, IL-1β and TNF-α than those in Con. (4) Cb and CD had lower caecal acetic and propionic content than the Anti group (p < 0.05). (5) The community richness of Con was significantly higher than that of Anti (p < 0.05). The relative abundance of Alistipes and Ruminococcaceae-UCG-014 in Cb and CD supplemented birds were lower than those in Con (p < 0.05). The relative abundant of Escherichia-Shigella in CD was higher than Con and Anti (p < 0.05). Conclusions: These data indicated that dietary C. butyricum and 1, 25-dihydroxyvitamin D 3 can improve the growth performance, immunity responses, antioxidation, bone development and intestinal microflora in early-stage broilers. Significance and Impact of the Study: Oral administration of C. butyricum or C. butyricum combined with 1,25-dihydroxyvitamin D 3 enhanced immunity and antioxidant activity in early-stage birds.