BACKGROUND: Targeting cancer stem cells (CSCs)-mediated aggressive features of non-small cell lung cancer (NSCLC) is a promising anticancer approach. This can be accomplished via suppressing critical mediators, such as functional mitochondria, aldehyde dehydrogenase (ALDH)1A, and telomere protectors (telomerase reverse transcriptase (TERT) and telomere repeat binding factor (TRF)1). MATERIALS & METHODS: Copper nanocomplexes (diethyldithiocarbamate (DE)-CuO nanoparticles (NPs) and DE-Cu NPs) were prepared using the simplest green chemistry method and assessed for inducing mitochondrial dysfunction-dependent non-apoptotic pathway (cuproptosis) and repressing CSC markers. RESULTS: DE-CuO NPs had higher growth inhibition for NSCLC (A549, H520, and H1299) spheroids than DE-Cu NPs. DE-CuO NPs had higher uptake rate and prooxidant effect resulting in lower mitochondrial membrane potential and mitochondrial DNA copy number, as well as stronger inhibition of telomerase and ALDH1A than DE-Cu NPs. This caused dramatic redox imbalance and lowering AKT pathway (activator of telomere stabilizers and stemness)-mediated repression of TERT and TRF1 protein levels as well as phosphorylated NF-κB subunit (p65) led to collapsing telomeres, as evidenced by downregulating TERT regulators and confocal microscopy. In animal study, this active nanocomplex revealed powerful and selective therapeutic tumor-targeting effects, with no evidence of toxicity to healthy tissues. CONCLUSION: DE-CuO nanocomplex is deemed as promising nanomedicine for NSCLC.