Seventy-two piglets aged at 25 d were chosen to investigate the effects of Bacillus subtilis DSM32315 supplementation in diets with different protein levels on growth performance, intestinal barrier function, and gut microbiota profile in a 42-d trial. The animals were allotted to four treatment groups in a randomized complete block design involving a 2 (protein levels) × 2 (probiotic levels) factorial arrangement of treatments. Two protein levels included the high CP (HP) diets (0 to 14 d, 20.5%; 15 to 42 d, 19.5%) and the low CP (LP) diets (0 to 14 d, 18%; 15 to 42 d, 17%), and added probiotic (PRO) levels included at 0 and 500 mg/kg diet. Two interactions between CP and PRO for ADG (P < 0.01) and F/G (P < 0.05) were observed in phase 1. Within the piglets given the LP diet, probiotic supplementation increased ADG and decreased F/G ratio. Likewise, there were interactions between CP and PRO on the digestibility of CP (P < 0.01) and EE (P < 0.05), and probiotic supplementation increased the digestibility of CP and ether extract (EE) of piglets fed with LP diet, but that was not the case for piglets fed with HP diet. Furthermore, there were interactions between CP and PRO on villus height (P < 0.01) and villus height:crypt depth ratio (P < 0.05) in ileum. Piglets fed with LP diet containing probiotic had the greatest villus height and villus height:crypt depth ratio in ileum among treatments. There were also main effects of PRO on the propionic acid (P < 0.05) and butyric acid (P < 0.05), and the concentrations of propionic acid and butyric acid in colonic digesta were increased with the inclusion of probiotic in diet. Piglets fed with LP diet containing probiotic had the greatest population of Bacillus and Bifidobacterium (P < 0.05) in colon. In addition, there were interactions between CP and PRO on the mRNA expressions of occludin-1 (P < 0.05), epidermal growth factor (EGF) (P < 0.05), and insulin-like growth factor 1 receptor (IGF-1R) (P < 0.05). The LP fed piglets plus probiotic exhibited the greatest mRNA expressions of occludin-1, EGF, and IGF-1R in ileum compared with other treatments. In conclusion, moderate dietary protein restriction combining with the addition of B. subtilis DSM32315 synergistically increased growth performance, altered hindgut bacterial composition and metabolites, maintained intestinal barrier function in ileum of piglets.