Abstract:[Background] Infection with Clostridium perfringens causes necrotizing enteritis in animals, which is commonly treated by antibiotics. With the limitation of antibiotics in feed and husbandry in China, probiotics and its metabolites have become the focus of current research. [Objective] This study was conducted to reveal the properties of B. subtilis BS-2 against Clostridium perfringens. [Methods] The growth curve of B. subtilis BS-2, the antimicrobial properties of its metabolites and the mRNA levels of bacteriocin gene cluster were detected. [Results] The metabolites of B. subtilis BS-2 had no inhibitory effect on Gram-negative bacteria, whereas they had strong activity against Gram-positive bacteria. The activity of the strain against C. perfringens increased rapidly within 2–12 h and remained stable within 12–24 h. The antibacterial properties of the metabolites were not affected by pepsin, trypsin or protease K, which demonstrated good thermal stability. The mRNA levels of bacteriocin gene cluster showed that the inhibitory activity of B. subtilis BS-2 against C. perfringens was related to the expression of surfactin and mersacidin. [Conclusion] B. subtilis BS-2 may inhibit C. perfringens by regulating the expression of surfactin and mersacidin.