The low efficiency of endosomal escape has been considered a bottleneck for the cytosolic delivery of biomacromolecules such as proteins and DNA. Although fusogenic peptides (FPs) such as HA2 have been employed to improve the intracellular delivery of biomacromolecules, the FPs studied thus far are not adequately efficient in enabling endosomal escape; therefore, novel FPs with higher activity are required. In this context, we focused on FPs derived from a sea urchin fertilization protein, bindin, which is involved in gamete recognition (B18, residues 103-120 and B55, residues 83-137 of mature bindin). We show that enhanced green fluorescent protein (EGFP)-fused B55 peptide binds to plasma membranes more strongly than EGFP-B18 and promotes the intracellular delivery of dextrans, which were co-administered using the trans method in a pH-dependent manner without affecting cell viability and proliferation, whereas conventional EGFP-HA2 did not affect dextran internalization. Furthermore, EGFP-B55 promoted the intracellular delivery of biomacromolecules such as antibodies, ribonuclease and plasmidic DNA using the trans method. Because the promotion of intracellular delivery by EGFP-B55 was suppressed by endocytosis inhibitors, EGFP-B55 is considered to have facilitated the endosomal escape of co-administered cargos. These results suggested that an FP that promotes the intracellular delivery of a variety of biomacromolecules with no detectable cytotoxicity should be useful for the cytosolic delivery of membrane-impermeable molecules for biomedical and biotechnological applications.