A novel paradigm in tumor biology suggests that non-small cell lung cancer (NSCLC) growth is driven by lung cancer stem cell-like cells (LCSCs), but here are still not any effective strategies to remove LCSCs. The bispecific antibody is a novel antibody, which can target two different antigens and mediate specific killing effects by selectively redirecting effector cells to the target cells. Here, we designed and synthesized a new bispecific antibody (BsAb), BsAb-5, that can target cellular-mesenchymal to epithelial transition factor (c-MET) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) in CD166+ LCSCs with high affinity and specificity, for the first time. We showed that BsAb-5 could inhibit hepatocyte growth factor (HGF)-mediated tumor development, including proliferation, migration, and apoptosis, serving as an inhibitory c-MET antibody. Moreover, we demonstrated that mechanisms responsible for BsAb-5 in CD166+ LCSCs included inducing c-MET degradation and inhibition HGF-stimulated c-MET-Notch pathway by using AdHGF infection, nuclei location and western-blot assays. In vivo, xenograft analysis revealed that mice on BsAb-5 group showed significantly reduced tumor volume. At the meantime, the observed anti-tumor effects of BsAb-5 were dependent on considerably suppressing Tregs and up-regulating effector T cells. On the basis of these results, we have identified a potential bispecific antibody drug, which can effectively target c-MET and CTLA-4 in CD166+ LCSCs for the treatment of human NSCLC.