A series of star-shaped cationic polymers, termed star vectors (SVs), has been developed as effective nonviral gene delivery carriers. In this study, we separated SVs into several fractions having different molecular weights with very narrow molecular weight distributions in order to examine in detail the influence of the molecular weight of the SVs on the gene transfection efficiency. As a model compound for several types of SVs, 4-branched poly(N,N-dimethylaminopropyl acrylamide) having a molecular weight (M(n)) of approximately 35 kDa and polydispersity of 1.6 was prepared by iniferter-based radical polymerization. The SVs were separated using size-exclusion chromatography to obtain seven fractions having M(n) ranging from 27 kDa to 73 kDa with polydispersity ranging from 1.1 to 1.2. All the fractionated SVs have similar pH of 10.2-10.4 and were able to interact with and condense luciferase-encoding plasmid deoxyribonucleic acid (DNA) to yield SV/DNA polyplexes. A water-soluble tetrazolium-1 (WST) assay showed that all SVs had minimal cellular cytotoxicity under an N/P charge ratio of 10. The critical micellar concentration decreased with an increase in the M(n) of the fractionated SVs; however, the particle size of the polyplexes, exclusion activity of ethidium bromide, and zeta-potential of the polyplexes increased. An in vitro evaluation using COS-1 cells at an N/P ratio of 10 showed that transfection activity increased almost linearly with M(n). The highest transfection activity was obtained for SVs with the highest M(n) (73 kDa), which was over 7 times that for the SVs with the lowest M(n) (27 kDa), the nonfractionated original SV, or PEI standard. The transfection efficiency was more correlated with the amphiphilicity or hydrophobicity of the SVs and the surface potential and condensate density of the polyplexes than with the particle size.