Direct delivery of enzymes into intact plants using cell-penetrating peptides (CPPs) is an attractive approach for modifying plant functions without genetic modification. However, by conventional methods, it is difficult to maintain the enzyme activity for a long time because of proteolysis of the enzymes under physiological conditions. Here, we developed a novel enzyme delivery system using polyion complex vesicles (PICsomes) to protect the enzyme from proteases. We created PICsome-bearing reactive groups at the surface by mixing an anionic block copolymer, alkyne-TEG-P(Lys-COOH), and a cationic peptide, P(Lys). The PICsome encapsulated neomycin phosphotransferase II (NPTII), a kanamycin resistance enzyme, and protected NPTII from proteases in vitro. A CPP-modified PICsome delivered NPTII into the root hair cells of Arabidopsis thaliana seedlings and provided kanamycin resistance in the seedlings that lasted for 7 days. Thus, the PICsome-mediated enzyme delivery system is a promising method for imparting long-term transient traits to plants without genetic modification.