Intramuscular gene delivery materials are of great importance in plasmid-based gene therapy system, but there is limited information so far on how to design and synthesize them. A previous study showed that the peptide dendron-based triblock copolymer with its components arranged in a reversed biomembrane architecture could significantly increase intramuscular gene delivery and expression. Herein, we wonder whether copolymers with biomembrane-mimicking arrangement may have similar function on intramuscular gene delivery. Meanwhile, it is of great significance to uncover the influence of electric charge and molecular structure on the function of the copolymers. To address the issues, amphiphilic triblock copolymers arranged in hydrophilic-hydrophobic-hydrophilic structure were constructed despite the paradoxical characteristics and difficulties in synthesizing such hydrophilic but electroneutral molecules. The as-prepared two copolymers, dendronG2(L-lysine-OH)-poly propylene glycol(2k)(PPG(2k))-dendronG2(L-lysine-OH) (rL(2)PL(2)) and dendronG3(L-lysine-OH)-PPG(2k)-dendronG3(L-lysine-OH) (rL(3)PL(3)), were in similar structure but had different hydrophilic components and surface charges, thus leading to different capabilities in gene delivery and expression in skeletal muscle. rL(2)PL(2) was more efficient than Pluronic L64 and rL(3)PL(3) when mediating luciferase, beta-galactosidase, and fluorescent protein expressions. Furthermore, rL(2)PL(2)-mediated growth-hormone-releasing hormone expression could significantly induce mouse body weight increase in the first 21 days after injection. In addition, both rL(2)PL(2) and rL(3)PL(3) showed good in vivo biosafety in local and systemic administration. Altogether, rL(2)PL(2)-mediated gene expression in skeletal muscle exhibited applicable potential for gene therapy. The study revealed that the molecular structure and electric charge were critical factors governing the function of the copolymers for intramuscular gene delivery. It can be concluded that, combined with the previous study, both structural arrangements either reverse or similar to the biomembrane are effective in designing such copolymers. It also provides an innovative way in designing and synthesizing new electroneutralized triblock copolymers, which could be used safely and efficiently for intramuscular gene delivery.