Objective Platinum-based chemotherapy is the first-line treatment for non–small cell lung cancer, but the chemoresistance of tumor cells continues to be a considerable challenge in the management of NSCLCs, leading to recurrence of most patients. CD133 (prominin-1) is a five-transmembrane glycoprotein, and recent evidence suggests that CD133+ cells are the cause of drug resistance and tumor recurrence. In this study, the correlation between cisplatin and CD133+ cells was investigated systematically.Methods Four lung cancer cell lines, including A549, H460, 801D and H1299, were treated with different concentrations of cisplatin. Cell viability was determined by MTT assay. Sphere-forming assay was performed to detect the capability of sphere-forming. CD133+ cells was detected by BD FACScaliber flow cytometer. Results The results showed that cisplatin could increase the number of CD133+ cells in both time- and dose-dependent manner. The enrichment would weaken but the proportion of CD133+ cells was still higher than the basic level as incubation time extended after cisplatin was withdrawn. Compared with adherent culture, the proportion of CD133+ cells was higher when the cells were maintained suspension culture. The proportion of CD133+ cells significantly increased when cisplatin was provided in suspension culture. Conclusion These results revealed that cisplatin induces the enrichment of CD133+ cells and CD133 is a new therapeutic target. Our data partially explained drug resistance to second-line chemotherapy in cisplatin-treated patients with NSCLCs.