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Self-assembled DNA nanostructures hold great promise in biosensing, drug delivery and nanomedicine. Nevertheless, challenges like instability and inefficiency in cellular uptake of DNA nanostructures under physiological conditions limit their practical use. To tackle these obstacles, this study proposes a novel approach that integrates the cationic polymer polyethyleneimine (PEI) with DNA self-assembly. The hypothesis is that the positively charged linear PEI can facilitate the self-assembly of DNA nanostructures, safeguard them against harsh conditions and impart them with the cellular penetration characteristic of PEI. As a demonstration, a DNA nanotube (PNT) was successfully synthesized through PEI mediation, and it exhibited significantly enhanced stability and cellular uptake efficiency compared to conventional Mg2+-assembled DNA nanotubes. The internalization mechanism was further found to be both clathrin-mediated and caveolin-mediated endocytosis, influenced by both PEI and DNA. To showcase the applicability of this hybrid nanostructure for biomedical settings, the KRAS siRNA-loaded PNT was efficiently delivered into lung adenocarcinoma cells, leading to excellent anticancer effects in vitro. These findings suggest that the PEI-mediated DNA assembly could become a valuable tool for future biomedical applications. [ABSTRACT FROM AUTHOR]