Lumichrome, a major derivative of riboflavin, may exhibit pharmacological activity against cancer cells. Riboflavin is a vitamin found in food, however, certain evidence has suggested its possible potentiating effects on cancer progression. Here, we have shown for the first time that unlike riboflavin, lumichrome can suppress lung cancer cell growth and reduce survival in both normal and anchorage-independent conditions. In addition, lumichrome induced apoptosis in lung cancer cells via a p53-dependent mitochondrial mechanism with substantial selectivity, shown by its lesser toxicity to the normal primary dermal papilla cells. The potency of lumichrome in killing lung cancer cells was found to be comparable to that of cisplatin, a standard chemotherapeutic drug for lung cancer treatment. With regard to the mechanism, lumichrome significantly upregulated p53 and decreased its downstream target BCL-2. Such a shift of BCL-2 family protein balance further activated caspase-9 and -3 and finally executed apoptosis. Furthermore, lumichrome potentially suppressed cancer stem cells (CSCs) in lung cancer by dramatically suppressing CSC markers together with the CSC-maintaining cell signaling namely protein kinase B (AKT) and β-catenin. To conclude, the present study has unraveled a novel role and mechanism of lumichrome against lung cancer that may benefit the development of the compound for management of the disease.