One of the critical challenges for efficient non-viral gene delivery in vivo is the ability to control the transport properties in biological milieu of DNA-containing nanoparticles. Recently, nanoparticle shape has been identified as an important factor determining these properties. However, until now it has not been possible to control the shape of nanoparticles containing packaged plasmid DNA. We have developed a new approach to achieve effective shape control of nanocomplexes of plasmid DNA and polyethylene glycol (PEG)–polycation copolymers. Specifically, we have developed the experimental strategies to realize shape tunability from spherical and rod-like to worm-like DNA/polymer nanoparticles through variation of polymer structure and solvent polarity, and molecular dynamics simulations aiming at identifying the key parameters modulating shape control in DNA/polymer nanoparticle assembly. In addition, we have also developed methods to characterize the composition and its distribution of these complex nanoparticles. More importantly, we have demonstrated the shape-dependent cellular update, transfection efficiency in vitro and in vivo. These findings open up a new avenue for controlling the shape of DNA-compacting nanoparticles and enhancing gene delivery efficiency. These micelles may serve as virus-mimetic nanoparticles for elucidating the role of shape in determining particle transport properties and bioactivities.