<p>The first-line chemical drug cisplatin used to treat the non-small cell lung cancer (NSCLC) often becomes ineffective due to acquired drug resistance of cancer cells. This effect and related cancer progression are related to the presence of cancer stem cells in the tumor. Tyrosine kinase Src is responsible for the appearance and development of cancer stem cells. Here, we examined the effects of Src on the oncogenic properties of cisplatin-resistant NSCLC cell lines (H358R and A549R) and the effect of inhibition of this tyrosine kinase on the sensitivity of cancer cells to cisplatin by Western blotting and immunofluorescence staining as well as migration, invasion, sphere colony, and clone formation assays. In H358R and A549R cells, the levels of phosphorylated kinase Src (pSrc) and stemness marker CD133 were elevated in comparison with the parental ones. The cisplatin-resistant cells demonstrated increased self-renewal ability and formed significantly bigger tumor spheres than their parental cells. Inhibition of Src with its inhibitor CGP77675 attenuated stemness of H358R and A549R cells; moreover, it enhanced the inhibitory effects of cisplatin on cell proliferation, migration, and invasion. The data indicated that Src-induced stemness plays an important role in developed resistance of NSCLC cells to cisplatin. Inhibition of Src attenuated stemness and acquired resistance to cisplatin, which can be beneficial for treating the cisplatin-resistant NSCLC.</p>