Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide with potent cytotoxic activity against cancer cells. The antimicrobial activity of LfcinB resides in its RRWQWR amino acid sequence (referred to here as LfcinB6); however, the anticancer activity of LfcinB6 is not known. Here, we show that free LfcinB6 did not kill T-leukemia or breast cancer cells but LfcinB6 was strongly cytotoxic when delivered to the cytosolic compartment by fusogenic liposomes. LfcinB6 bound weakly to isolated mitochondria but, unlike LfcinB, did not permeabilize mitochondria or cause cytochrome c to be released. Cathepsin B and caspase activity were important for cytotoxicity caused by intracellular LfcinB6 whereas reactive oxygen species were not involved. The mechanism of LfcinB6-induced cytotoxicity is therefore different from that of LfcinB. We suggest that LfcinB6, in combination with a fusogenic liposome delivery system that selectively targets malignant cells, has potential as a novel anticancer agent.