To investigate the effects of Clostridium butyricum on serum biochemical indices, antioxidant function and intestinal microbiota in weaned piglets, a total of 360 25-day-old healthy“Duroc×Landrace×Yorkshire” weaned piglets with the average body mass of (6.24±0.32) kg were selected and randomly divided into five groups. Each group had four replicates with 18 piglets per replicate. Piglets in the control group (CG) were fed with a corn-soybean type basal diet, and those in the experimental groups (EG) were fed with experimental diet, which added 250, 500, 1 000 and 2 000 mg/kg Clostridium butyricum in basal diet for 30 days, respectively. The results showed as follows: 1) No significant difference was found between the CG and EG on the serum biochemical indices such as glutamic oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT), blood urea nitrogen (BUN) and alkaline phosphatase (AKP) (P＞0.05). Compared with the CG, the serum total protein (TP) level in the EG was increased by 10.45% (P＞0.05), 17.64% (P＜0.05), 15.24% (P＜0.05) and 17.54% (P＜0.05), respectively; the concentrations of serum albumin (ALB) in the EG were increased by 0.78% (P＞0.05), 3.80% (P＞0.05), 3.50% (P＞0.05) and 5.52% (P＜0.05), respectively. 2) The concentration of serum catalase (CAT) in the 1 000 mg/kg group was increased by 65.87% (P＜0.05). The total antioxidant capacity (TAOC) level of liver in the 500 mg/kg group was increased by 13.14% (P＜0.05). 3) The diet supplemented with Clostridium butyricum significantly decreased the number of Escherichia coli and Salmonella in the cecum of weaned piglets (P＜0.05 or P＜0.01). Compared with the CG, the number of Lactobacillus and Bifidobacterium in the EG increased at different degrees (P＞0.05). The results indicate that the diet supplemented with Clostridium butyricum can alleviate oxidative stress during weaning and inhibit the growth of harmful intestinal bacteria of weaned piglets. The optimal dietary Clostridium butyricum of weaned piglets at the age of 25 to 55 days is 500 mg/kg.