In an attempt to demonstrate the efficacy of Bacillus subtilis PB6, isolated from the gut of a healthy chicken, on broiler performance, an experimental trial was conducted in which broilers, infected or uninfected with a pathogenic strain of Escherichia coli, were supplemented with B. subtilis PB6 (CloSTAT) or zinc bacitracin-colistin sulfate and compared against negative controls given no antibiotic supplementation. We observed 10- and 8-point improvements in feed conversion ratio (FCR) in 42-d-old uninfected broilers treated with B. subtilis PB6 when compared with the negative and antibiotic controls, respectively. However, infected birds supplemented with B. subtilis PB6 registered a significant 15-point FCR improvement over those in the negative control group (P < 0.05). Compared with the negative control, the increase in body weights of uninfected and infected 42-d-old broilers receiving B. subtilis PB6 were 97 and 152 g, respectively (P < 0.05). The growth-promoting and protective results from this study indicated that B. subtilis PB6 not only helped in the maintenance of beneficial bacteria but also could act as a replacement for antimicrobial growth promoters in broilers. The percentages of mortality of infected birds within antibiotic-treated groups and B. subtilis PB6-treated groups were reduced from 14% to 6 and 8%, respectively. Numerically, uninfected birds supplemented with B. subtilis PB6 had elevated levels of lactobacilli (1.4 to 4.5 × 107 cfu/g) in their intestinal tracts (32- and 42-d-old broilers) compared with the controls. Even after being challenged with a pathogenic strain of E. coli, the lactobacilli counts in the B. subtilis PB6-treated birds tended to remain the same as those receiving antibiotic and were considerably higher than those of the untreated birds.