Non-small cell lung cancer (NSCLC) ranks among the most prevalent malignancies globally. Gboxin, a novel inhibitor of mitochondrial complex V that exerts unique anti-tumor effects via oxidative phosphorylation inhibition, but shows no efficacy against NSCLC in vivo. Through chemical structure optimization, we designed and synthesized Gboxin analog Y9, which demonstrates significantly enhanced potency over its predecessor. Specifically, Y9 inhibited NSCLC significantly more strongly than Gboxin and possessed the ability to inhibit cell cycle progression and induce oxidative stress similar to Gboxin. Further investigation revealed that unlike Gboxin, Y9 selectively acidifies lysosomes and induces lysosomal dysfunction. This leads to hyperactive autophagy with impaired substrate clearance, and ultimately resulting in apoptosis. Animal studies confirmed the efficacy of Y9 in suppressing tumor growth in a xenograft mouse model. Collectively, Y9 is a distinctive Gboxin analog that outperforms its prototype by inducing lysosomal dysfunction and apoptosis, and has the potential to be developed as a novel anti-NSCLC lead compound.