We separated CSCs from cell lines and lung cancer tissues by CSC marker CD133 and ALDH. Then we knock down the expression of TLR9 and then investigate biological changes of CSCs, including sphere formation, self-renewing ability, invasion, resistance to drugs, and in vivo tumor formation. Result: We found toll-like receptor (TLR9) are constitutively overexpressed on cancer stem cells (CSCs) derived from lung cancer cell lines and patients' tissue when compared with non-stem cells. Knocking down of TLR9 in lung CSCs resulted in inhibited capacity for proliferation, invasion, tumorigenesis and resistance to chemotherapeutic drugs. Furthermore, cell cycle was arrested at G2/M stages and more apoptosis existed after TLR9 expression decreased. Further analysis indicated that TLR9 maintain the stemness of cancer cells by changing expression of other stem cell markers Gli1, Notch1 and beta-catenin, drug transporters ABCB1, ABCC1 and ABCG2 and anti-apoptotic factors BCL2 and Survivin. Of which, beta-catenin, was found to be most obviously related with TLR9 expression. Moreover, TLR9 expression was positively associated with tumor differentiation and inversely associated with lung cancer patient survival in clinic. Conclusion: Based on above data, TLR9 is crucial for marker and phenotype of lung CSC. It might work as a lung cancer stem cell maintainer.