The lack of safe and effective carriers for RNA interference therapeutics remains a barrier for its wide clinical application. In this study, guanidino groups were incorporated into poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly(L-lysine) (mPEG-b-PCL-b-PLL, AG0) by simple replacement of the amino groups on PLL segments by the guanidino groups to enhance the transfection performance by mimicking the transmembrane function of cell penetrating peptides, such as TAT or other arginine-rich peptides. The guanidinated copolymers (AG1–AG3) displayed similar siRNA-binding capacity to AG0, but less cytotoxicity and higher silencing efficiency than AG0. Typically, AG3 with full replacement of the amino groups by guanidino groups exhibited higher silencing efficiency than PEI-25k and Lipofectamine 2000. Cell uptake and cell imaging experiments showed that the enhanced silencing efficiency of AG3–siRNA complex was due to the enhanced endocytosis cross the cell-membrane and the enhanced escape from the endosomes/lyosomes. The guanidino groups on the polylysine units were responsible for these enhancements although they are attached to the polymer backbone with a spacer of (CH₂)₄, in comparison with (CH₂)₃ in polyarginine. In conclusion, guanidination of mPEG-b-PCL-b-PLL resulted in a less toxic and more efficient siRNA vector and contribution of the guanidine groups to cell-membrane penetration and endosome/lyosome-membrane penetration was demonstrated. Therefore, replacement of the amino groups in conventional gene delivery vectors with guanidine groups might be a useful strategy of developing novel gene or drug delivery vectors.