Brefeldin A (BFA) is an fungal antibiotic that induces ER stress in eukaryotic cells. Cancer stem cells (CSCs) have indefinite potential for self-renewal and the capability to drive tumorigenesis. The effects of BFA on CSCs have not been fully investigated. In this article we investigated the inhibitory effects of BFA on human lung cancer A549 and H1299 cells. Cytotoxicity toward suspension cultures has been used as a method for the preliminary screening of drugs targeting CSCs. We showed that BFA effectively reduced the survival of suspension A549 cells (EC50 =˜30 ng/ml) by inducing apoptosis, and inhibited the clonogenic activity of A549 and H1299 CSCs at the same concentration range. We also demonstrated that BFA reduced the migration ability of A549 and H1299 cells. These results suggest that BFA can effectively suppress the progression of lung cancer during tumorigenesis and metastasis. The results of this study may help develop novel therapy for future treatment of lung cancer.