Owing to their diverse functions and tunable physicochemical properties, peptides are promising alternatives to the conventional gene delivery tools that are available for plant systems. However, peptide-mediated gene delivery is limited by low transfection efficiency in plants because of the insufficient cytosolic translocation of DNA cargo. Here, we report a dual peptide-based gene delivery system for the efficient transfection of plant callus cells. This system is based on the combination of an artificial peptide composed of cationic cell-penetrating and hydrophobic endosomal escape domains with a gene carrier peptide composed of amphiphilic cell-penetrating and cationic DNA-binding domains. Cellular internalization and transfection studies revealed that this dual peptide-based system enables more efficient transfection of callus cells than does a carrier peptide alone by enhancing the endocytic uptake and subsequent cytosolic translocation of a carrier peptide/DNA complex. The present strategy will expand the utility of peptide-mediated plant gene delivery for a wide range of applications and basic research.