Bacillus subtilis is popularly used as a probiotic in many fields. Although recent research has found that some secondary metabolites of B. subtilis could cause hemolysis, the hemolytic mechanism in B. subtilis is still unclear. In this paper, the hemolysis-associated gene ytjA was cloned and expressed in Escherichia coli, and the hemolytic activity of the expressed soluble protein was indicated by the presence of clear hemolytic zones on sheep blood agar plates. In addition, reverse transcriptase-polymerase chain reaction analysis confirmed that the ytjA gene was transcribed in B. subtilis. These results suggest that ytjA is one of the hemolysin genes responsible for hemolysis in B. subtilis.