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Chemotherapy is regarded as a widely used and effective treatment strategy for lung cancer, although most conventional chemotherapeutics cause severe toxic side-effects due to their indiscriminate attacks on both cancerous and normal cells. Although nucleic acid nanomaterials are emerging as a promising drug delivery strategy, their clinical applications are limited by rapid degradation by nucleases and difficulties in targeting cancer cells. In this study, we have developed a Rhein-loaded aptamer-based DNA nanotube (DNT-S6@Rhein) for the targeted and efficient therapy of non-small cell lung cancer. Through the palindrome segments, two specified oligonucleotides were hybridized and folded into the well-defined nanotubes (DNT-S6), with the S6 aptamer distributed outside. The obtained nanotubes exhibited excellent serum stability and targeting ability towards A549 cells due to the firm structure and decoration of the S6 aptamer. Rhein, as an antitumor drug and DNA intercalator, can be effectively inserted into the DNT-S6. The drug-loaded nanotubes rapidly disassembled in intracellular environment and then the released Rhein was found to activate cellular apoptotic process and significantly suppress proliferation, migration and invasion of A549 cells. Moreover, DNT-S6@Rhein could efficiently accumulate in tumor regions, offering compelling therapeutic efficacy and biocompatibility under both in vitro and in vivo settings. These findings of this study provide a promising strategy for mitigating the inevitable systemic side-effects of chemotherapy and expand the potential application of DNA nanostructure on targeted drug delivery., Competing Interests: Compliance and ethics. The authors declare that they have no conflict of interest., (© 2024. Science China Press.)