Due to membrane impermeability of proteins, intracellular delivery of protein is of significant challenge. Although numerous protein carriers have been reported, it remains difficult to efficiently deliver proteins with different isoelectric points into cells. Herein, guanidinium perfunctionalized pillar[5]arene (GP5) was employed for efficient delivery of proteins with different isoelectric points into different cell lines, and the bioactivities of the proteins were well maintained after intracellular delivery. After comparison with linear cell-penetrating peptides, unsymmetrical guanidinium-macrocycles, primary and quaternary ammonium functionalized pillar[5]arene derivatives, and the monomer of GP5, the high protein delivery potency of GP5 was mainly attributed to the densely pre-organized guanidinium groups on both sides of the pillar[5]arene skeleton, which could not only facilitate GP5 to bind with proteins to form protein nano-aggregates, but also promote cellular uptake of proteins via interactions with cellular surface. This study offers important new insights to the design and development of cell-penetrating peptide mimetic molecules for protein transductions.