Chemoresistance is one of the most important causes of ovarian cancer‑related deaths. Recently, cancer stem cells (CSCs) have been recognized as the source of chemoresistance in ovarian cancer. However, the underlying mechanisms that regulate the chemoresistance of ovarian CSCs (OCSCs) remain unclear. The aim of the present study was to investigate the roles of S100B in the regulation of OCSC chemoresistance, which provides a novel therapeutic target. We observed high expression of S100B in CD133+ OCSCs derived from ovarian cancer cell lines and primary tumors and in cisplatin‑resistant patient samples. Then, we determined that S100B knockdown promoted the apoptosis of OCSCs after treatment with different concentrations of cisplatin. The underlying mechanism of S100B‑mediated chemoresistance in OCSCs may be through p53 inhibition. Furthermore, drug‑resistance genes, including MDR1 and MRP1, were involved in the process of S100B‑mediated OCSC chemoresistance. In conclusion, our results elucidated the importance of S100B in the maintenance of OCSC chemoresistance, which may provide a promising therapeutic target for ovarian cancer.