The present study was conducted to investigate effects of Bacillus subtilis on growth performance, serum parameters, digestive enzymes, intestinal morphology, and colonic microbiota in piglets. A total of 72 piglets were weighed and randomly allotted into three treatments (four replication pens per treatment with six piglets/pen) for a 28-day experiment. The dietary treatments were as follows: basal diet (control group, CTR), basal diet supplementation with antibiotic (antibiotic group, ABT), and basal diet supplementation with 0.1% Bacillus subtilis (probiotic group, PBT). The average daily gain of body weight increased in both the ABT and PBT groups, and dietary antibiotics decreased the feed:gain ratio (F:G), as compared to the CTR group (P < 0.05). Both ABT and PBT piglets had increased serum triglycerides and lipase, amylase, maltase activities and villus height:crypt depth ratio (V/C) in ileum (P < 0.05). The PBT group also showed an increase in serum glucose and villus height in the ileum (P < 0.05). Dietary antibiotics increased Lactobacillus johnsonii, as compared to the CTR group, but decreased bacterial diversity and increased Escherichia coli, as compared to the PBT group (P < 0.05). Piglets dietary with B. subtilis modulated the microbiota by increasing the abundance of Firmicutes (L. johnsonii, L. reuteri) and decreasing the abundance of E. coli, as compared to the control group (P < 0.05). These results indicate that dietary of B. subtilis improves growth performance and intestinal health and can be a promising alternative to antibiotics in piglets diet.