Novel star-shaped copolymers consisting of multiarm polyethylene glycol and low molecular weight linear polyethylenimines (MAPEG-LPEIs) with a high transfection efficiency and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. The cationic polymers were prepared by conjugating low molecular weight linear PEI (2.5 kDa) to six-arm PEG-NHS (10 kDa) in two different compositions. Two copolymers, MAPEG-LPEI(3) and MAPEG-LPEI(6) with molecular weights of 17.5 kDa and 25 kDa respectively, were synthesized. The MAPEG-LPEI(3)/pDNA and MAPEG-LPEI(6)/pDNA polyplexes are stably dispersed in aqueous media with a narrowly distributed size range of <200 nm as determined by dynamic light scattering. Furthermore, these polyplexes showed different surface charges depending upon the relative proportion of MAPEG and LPEI. Moreover, these polyplexes can protect pDNA from enzymatic degradation in serum containing media up to 24 h. These polyplexes were able to efficiently transfect luciferase-coded reporter gene into HeLa cancer cells and showed considerable gene transfection efficacy even in 50% serum-conditioned media in vitro. MAPEG-LPEI(6) exhibited higher transfection activity than that of MAPEG-LPEI(3) at the same weight ratios. Furthermore, MAPEG-LPEI/pDNA polyplexes were less toxic than LPEI/pDNA complexes as determined by MTT assay. These favorable results could be attributed to the combined effect of low molecular weight LPEI and multiarm PEG. The special structural features of the multiarm star-shaped central PEG core play an important role in achieving higher transfection efficiency as it imparts higher charge density to polyplexes and prevents the unwanted aggregation of the smaller polyplex particles. These two important factors contributed toward enhanced gene transfection. On the other hand, LPEI provides low cytotoxicity and effective complexation with pDNA in the designed architecture. Therefore it is possible to achieve enhanced gene transfection by using these two components, namely, pivotal multiarm PEG core and LPEI, in optimal ratio as observed in the case of MAPEG-LPEI(6).