The major goal of this study is to design, synthesize, and evaluate linoleic acid and penetratin dual-functionalized chitosan (CS-Lin-Pen) as a nonviral gene carrier. The amphiphilic CS-Lin-Pen self-assembles to form cationic micelles in an aqueous environment. These polymeric micelles exhibited excellent hemocompatibility and cell viability, as evaluated by in vitro hemolysis and MTT assay, respectively. When CS-Lin-Pen micelles were added to plasmid DNA (pDNA) solution, the electrostatic interaction between the cationic micelles and anionic pDNA led to the formation of stable CS-Lin-Pen/pDNA polyplexes with ∼100 nm in size. The resultant polyplexes demonstrated ∼5-fold higher cellular uptake as compared to unmodified chitosan. Furthermore, CS-Lin-Pen micelles showed efficient protection of pDNA from DNase I attack and exhibited ∼34-40-fold higher transfection in comparison with unmodified chitosan in HEK 293, CHO, and HeLa cells. These findings illustrate that the CS-Lin-Pen micelles could be exploited as a potential nonviral vector for efficient gene therapy.