N-Isopropylacrylamide and allylamine copolymers (PNIALM) were prepared by radical polymerization method. To endow them with arginine-like cell membrane penetrating function, the aminos in PNIALMs were transformed to guanidinium groups by chemical modification. The formation and guanidinylation PNIALM were confirmed by NMR; the composition of copolymers and the degree of substitution of guanidino in modified copolymer (PNIALM-G) were estimated as well. The electrophoretic assay revealed that PNIALM-G was capable of condensing DNA in spite of lower binding affinity compared to its parent copolymers. The results of particle size analyzer and TEM indicated that at higher copolymer/DNA weight ratios, the copolymer/DNA complexes were condensed to nanoparticles. PNIALM-G1-3 was shown to be very efficient in mediating plasmid DNA transfection to COS-1 cells both in the presence and absence of serum, even superior to PEI.