Glioma-associated oncogene homolog 1 (GLI1), an oncogenic molecule in non-small cell lung cancer (NSCLC), promotes the growth of NSCLC by enhancing lung cancer stem cells (LCSCs). However, the mechanism responsible remains unknown. FOXP3 is known to maintain LCSCs. The aim of this study was to explore whether GLI1 enhanced LCSCs via stimulating FOXP3. Experiments were performed in NSCLC tissue samples, cell lines and the animal tumor model. The expression of GLI1- and LCSC-related molecules was assessed at protein and mRNA levels. Relevant cell functions were also determined. A tumor xenograft mouse model was established to confirm the oncogenic role of GLI1. We confirmed that the expression of GLI1 was up-regulated in the tumor tissues of NSCLC compared with adjacent non-tumor tissues. But no significant association between GLI1 and clinicopathological characteristics was found. GLI1 expression was positively correlated with FOXP3 and it could promote FOXP3 expression likely via acting on the promoter of FOXP3. Along with the upregulation of FOXP3, GLI1 increased the expression of LCSC markers, ALDH1A1 and OCT4A, and the formation of tumor spheres, whereas the inhibition of GLI1 decreased the above features. We also found the involvement of Notch1 activation in GLI1-mediated FOXP3 pathway. The In vivo mouse tumor model verified the positive role of GLI1 in the growth of the tumor. Collectively, this study has demonstrated that GLI1 stimulates FOXP3 to promote LCSCs. AJTR Copyright © 2020.