Abstract: Objective To explore the effect of Wnt/β-catenin signaling pathway on stemness maintenance in NCI-H23 cell. Methods The NCI-H23 was treated with the GSK-3β inhibitor （CHIR-99021） or β-catenin siRNA. Then， the MTT assay， stem cell culture， wound healing assay and Western blotting were used to detect proliferation ability， sphering ability and migration ability，respectively. Results Compared with control， there were increased proliferation ability and spher rate in NCI-H23 cell after treatment with CHIR-99021 for 24 h （P＜0.05）. Wound healing showed that the CH-treated cells were also faster than non-treatment. Meanwhile， CH-treated cells appeared to exhibit a declined level of p-GSK-3β. The expression of proliferating cell nuclear antigen，CD133，aldehyde dehydrogenase 1A1，Nanog and matrix metalloproteinase-2 showed a significantly enhance in both of NCI-H23 and NCI-H23 stem cell. After silencing β-catenin with β-catenin siRNA， the expression of CD133， ALDH1A1 and Nanog were declined and the sphere rate was decreased comparing with control. Conclusion Wnt/β-catenin signaling pathway involved in regulation of stemness in NCI-H23 cell.