Cancer stem cells (CSCs) are responsible for tumorigenesis and recurrence, so targeting CSCs is an effective method to potentially cure cancer. BTB/POZ domain-containing protein 7 (Btbd7) has been found in various cancers, including lung cancer and liver cancer, but the role of Btbd7 in non-small cell lung cancer (NSCLC), CSC self-renewal, and chemoresistance is still unknown. Therefore, in this study we found that the ratio of tumor sphere formation and stem cell transcription factors in CD133(+) cells was dramatically enhanced compared to parental cells, which indicated successful sorting of CD133(+) cells from A549. Meanwhile, Btbd7 and the markers of the epithelial mesenchymal transition (EMT) process were more highly expressed in CD133(+) cells than in parental cells. Silencing of Btbd7 significantly inhibited the self-renewal and EMT process in CD133(+) cells. Furthermore, we found that downregulation of Btbd7 promoted cell apoptosis and increased the sensitivity to paclitaxel in CD133(+) and parental cells. In conclusion, our results suggest that Btbd7 is a promising agent for the inhibition of survival and chemoresistance of cancer stem-like cells of NSCLC, which may act as an important therapeutic target in NSCLC.