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Due to the characteristics of high recurrence and metastasis, it is still difficult to cure lung cancer. Cancer stem cells (CSCs) are a group of tumor cells with self-renewal ability and differentiation potential, which are responsible for lung cancer recurrence. Therefore, targeting CSCs may provide a new strategy for lung cancer treatment. Thymoquinone (TQ), the main active ingredient isolated from black seed oil, has shown significant anti-cancer effects in various cancers. However, the effect of TQ on lung cancer stem cells (LCSCs) has never been clarified. In the present study, we successfully separated and enriched lung cancer tumorsphere cells. Our data showed that TQ significantly inhibited the stem-like properties of LCSCs. In addition, we found TQ promoted Yes-associated protein (YAP) phosphorylation and ubiquitination, and the inhibitory effects of TQ on LCSCs could be enhanced by silencing YAP. Taken together, these results suggest that TQ, functions by targeting YAP, may be a potential therapeutic agent against lung cancer. [ABSTRACT FROM AUTHOR]