It is reported that low concentration of amphiphilic triblock copolymers of pMeOx‐b‐pTHF‐b‐pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co‐injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.