Objective To explore the inhibitory effects of quercetin on the activity of A549 lung cancer stem cells. The role of quercetin on Wnt / β-catenin and Sonic Hedgehog SHH pathway was further illustrated. Method The lung cancer cell line A549 was cultured in vitro and treated with various concentrations of quercetin 0 5 10 25 50 100 and 200 μmol / L for 24 48 and 72 h cell vitality was detected by MTT assay. Lung cancer stem cells were isolated and enriched using sphere-forming assay. The effects of quercetin on the sphere formation and the expression of lung cancer stem cell specific markers were evaluated. The role of quercetin on the proliferation and apoptosis of sphere-forming cells was further determined by flow cytometry and western blot analysis. Finally the protein levels of key molecules of Wnt / β-catenin and SHH pathways were detected by western blot assay. Result Our results showed that quercetin significantly inhibited A549 cell viability in a time- dependent and concertation-dependent manner. Compared to the vehicle control group quercetin markedly suppressed the sphere formation ability of A549 cells and reduced the expression of lung cancer stem cell specific markers CD133 CD44 and aldehyde dehydrogenas 1A1 ALDH1A1 . Furthermore quercetin reduced proliferation and induced apoptosis of lung cancer stem cells. The levels of proliferating cell nuclear antigen PCNA Cyclin D1 and B-cell lymphoma- 2 Bcl2 were markedly downregulated while the expression of Bcl2-associated X Bax and cleaved cysteinyl aspartate specific proteinase Cleaved Caspase 3 was significantly upregulated upon quercetin treatment. The Wnt / β-catenin and SHH-related proteins phospho-glucogen synthase kinase-3β p-GSK3β Ser9 β-catenin c-Myc glioma-associated oncogene homolog 1 GLi1 GLi2 and SHH in lung cancer stem cells were also inhibited by quercetin. Conclusion Quercetin significantly suppressed the activity of lung cancer stem cells which might be associated with the downregulation of Wnt / β-catenin and SHH pathways.