Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-l-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P (nisA) ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.