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Ovarian cancer cells can form spheroids under serum‐free suspension culture conditions. The spheroids, which are enriched in cancer stem cells, can result in tumor dissemination and relapse. To identify new targetable molecules in ovarian cancer spheroids, we investigated the differential expression of genes in spheroids compared with that under monolayer culture conditions by  qPCR  microarray. We identified that  SOX 2 is overexpressed in spheroids. We then proved that  SOX 2 expression was increased in successive spheroid generations. Besides, knockdown of  SOX 2 expression in  SKOV 3 or  HO 8910 ovarian cancer spheroid cells decreased spheroid formation, cell proliferation, cell migration, resistance to Cisplatin treatment, tumorigenicity, and the expression of stemness‐related genes and epithelial to mesenchymal transition‐related genes, whereas overexpression of  SOX 2 in  SKOV 3 or  HO 8910 ovarian cancer cells showed the opposite effects. In addition, we found that  SOX 2 expression was closely associated with chemo‐resistance and poor prognosis in  EOC  patients. These results strongly suggest that  SOX 2 is required to maintain cancer stem cells in ovarian cancer. Targeting  SOX 2 in ovarian cancer may be therapeutically beneficial.