The aim of this study was to investigate the effect of HiWi gene silencing on lung cancer tumor stem cell proliferation and apoptosis using gene transfection and RNA interference. Moreover, we examined the feasibility of using the HiWi gene as a molecular target for the inhibition of lung cancer tumor stem cells (TSCs). shRNA eukaryotic expression vectors, pGenesil-2-HiWi1, pGenesil-2-HiWi2263 and pGenesil-2-control, targeting the HiWi gene were constructed. PBS served as the control group. The expression vector of the target HiWi gene shRNA was transfected into lung cancer TSCs with PEI as the medium. The conditions of lung cancer TSC proliferation and apoptosis in each group were examined using an MTT assay, fluorescence-activated cell sorting and Annexin V staining. The results showed that 24 h after transfection, the proliferation inhibition rates in the pGenesil-2-HiWi2263 (81.62%) and pGenesil-2-HiWi1 (73.16%) groups were higher as compared to the proliferation inhibition rate in the pGenesil-2-control group (8.54%). The apoptotic ratios in the pGenesil-2-HiWi1 and pGenesil-2-HiWi2263 groups were 26.16±1.21 and 28.06±1.78%, respectively, were higher as compared to those in the pGenesil-2-control group 2.86±0.09% (P<0.01). Our results suggest that HiWi gene silencing decreases proliferation and promotes apoptosis of lung cancer TSCs. Therefore, the HiWi gene could be used as a molecular target for the inhibition of the growth of lung cancer TSCs, which has potential value for the treatment of lung cancer.