The antineoplastic activity of host defense peptide Hymenochirin-1B, has been extensively studied. However, the mechanism still remains unknown. In this study, linear peptide, Hymenochirin-1B, was synthesized via solid-phase peptide synthesis and evaluated for its anticancer efficacy. We found Hymenochirin-1B induced lung cancer cell apoptosis and cell cycle arrest at the GO/G1 phase. Moreover, Hymenochirin-1B could enter the cells and colocalized with mitochondria. Furthermore, decrease of mitochondrial membrane potential, increase of reactive oxygen species and the expression of apoptosis-associated protein (Bax/Bcl-2 ratio and activated Caspase-3) were observed in NCI-H1299 and A549 cells after Hymenochirin-1B treatment, suggesting that Hymenochirin-1B induced apoptosis via mitochondrial pathway. Our results provide new insights on the anticancer mechanism of Hymenochirin-1B, which may contribute to its further development into an antineoplastic drug in the future. (C) 2019 Elsevier Inc. All rights reserved.