In the livestock feed industry, antibiotics are used to prevent disease, promote growth rate, and improve feed efficiency. However, antibiotic supplementation to animal feed results in increased bacterial resistance to antibiotics as well as antibiotic residues in animal products, which can negatively affect human health. Therefore, alternative sources of antibiotics are need- ed. Probiotics as an alternative to antibiotics in animal feed have been shown to increase feed efficiency and growth rate by improving microbial balance. Further, Bacillus sp. produces a wide spectrum of antibacte- rial peptides. The present study was conducted to investigate the effects of dietary supplementation with CS-32 on safety, growth rate, and feed efficiency. Antibacterial substance (5697.9 molecular weights) produced by CS-32 was isolated and purified from culture broth. Moreover, the results of minimal inhibi- tory concentration (MIC) test confirmed the excellent antibacterial effect of CS-32. In vivo, 0.1% and 1% CS-32 were fed to broiler chickens for 28 days. Feed efficiency was slightly higher in groups of chickens supplemented with 0.1% and 1% CS-32 than those of the control group. CS-32 had no significant effect on necropsy findings, hematology, or serum biochemistry, and there was no mortality. These results suggest that CS-32 among various biologically active substances may be safe and effective as a feed additive to improve growth rate and feed efficiency.