Buforin IIb is a novel cell-penetrating anticancer peptide derived from histone H2A. Here we analyzed the anticancer activity and cancer cell-killing mechanism of buforin IIb. Buforin IIb displayed selective cytotoxicity against 62 cancer cell lines by specifically targeting cancer cells through interaction with cell surface gangliosides. It traversed cancer cell membranes without damaging them and accumulated primarily in the nuclei. Once inside the cells, buforin IIb induced mitochondria-dependent apoptosis. In vivo analysis revealed that buforin IIb displayed significant tumor suppression activity in mice with tumor xenograft. Overall, these results suggest that buforin IIb constitutes a novel therapeutic agent for the treatment of cancers.