Background: To investigate the role of a topoisomerase I inhibitor (topotecan) in chemoresistant ovarian cancer-initiating cells. Materials and Methods: We isolated ovarian cancer-initiating cells (CP70 side-population cells) from the CP70 cell line using FACS Aria-based sorting and cultured them in suspension to form spheroids (CP70 side-population sphere [SPS]). Gene expression was assessed by microarray, to identify potentially effective chemotherapeutic drugs. An MTS assay was used to evaluate cell growth. Results: CP70 SPS cells showed significant resistance to the chemotherapeutic drugs cisplatin and paclitaxel. Microarray analysis demonstrated a high expression of topoisomerase-related genes in CP70 SPS cells. Topotecan inhibited ovarian cancer-initiating cells (CP70 SPS) in vitro more than it did their parental CP70 cells. This result was confirmed in tissues from human patients. Conclusions: Chemoresistant ovarian cancer-initiating cells exhibited high expression levels of topoisomerase, which could be an alternative target of adjuvant therapy for patients with chemoresistant ovarian cancer.