The study aimed to evaluate the effects of dietary Bacillus subtilis DSM 32540 supplementation with or without different fiber sources for piglets. A total of 150 piglets (7.20 ± 0.98 kg) weaned at 21 days old were assigned to one of five treatments in a randomized block design, with ten replicates and 3 piglets per pen. Treatments consisted of negative control (NC, without ZnO and amoxicillin), positive control (PC, with ZnO and amoxicillin), NC + 0.5 g/kg of probiotic (PRO), NC + 0.5 g/kg probiotic + 5 g/kg cellulose as IDF + 5 g/kg inulin as SDF (PROCI), and NC + 0.5 g/kg probiotic + 50 g/kg wheat bran (PROWB). From D0 to D14 and D0 to D28, piglets fed NC or PROWB had the lowest (P < 0.01) average daily gain (ADG) and final body weight (BW), while piglets fed PRO and PROCI had similar ADG and BW to those fed PC. From D0 to D14, piglets fed PRO and PC had lower (P < 0.01) feed conversion ratio (FCR) than those fed NC. From D0 to D28, piglets fed PROWB and NC had the highest (P < 0.01) FCR, while there was no difference in FCR among PC, PRO and PROCI groups. Piglets fed PRO, PROCI and PROWB had lower (P < 0.001) diarrhea incidence than those fed NC. Piglets fed PC, PRO and PROCI and those fed PRO and PROWB had higher (P < 0.01) duodenum and jejunum villus heights and villus to crypt ratios, respectively, than those fed NC. Piglets fed NC had the highest (P = 0.045) serum DAO content, and no difference was observed among groups PC, PRO, PROCI and PROWB on serum concentration of DAO. In conclusion, B. subtilis DSM 32,540 supplementation improves performance of piglets fed diets with or without pure fiber sources. In addition, regardless of the fiber source, B. subtilis DSM 32,540 reduces diarrhea incidence and improves intestinal morphology and integrity of nursery piglets.