Ovarian cancer is a highly malignant gynecological tumor that seriously endangers women's health. Previously, we demonstrated that anisomycin significantly inhibited the activity of ovarian cancer stem cells (OCSCs)in vitroandin vivo. In this study, anisomycin treatment of OCSCs significantly reduced the content of adenosine triphosphate and total glutathione, increased the extent of lipid peroxidation, and increased malondialdehyde, and Fe2+levels. The ferroptosis inhibitor Ferr-1 could significantly weaken the cytotoxicity of anisomycin. Subsequently, the cDNA microArray results suggested that anisomycin significantly reduced the transcription levels of gene clusters associated with protection from ferroptosis, such as those encoding members of the glutathione metabolism and autophagy signal transduction pathways. Bioinformatic analyses indicated that genes encoding core factors of these two pathways, and activating transcription factor 4 (ATF4), were significantly expressed in ovarian cancer tissues and correlated with poor prognosis. After overexpression or knockdown ofATF4, the ability of anisomycin to inhibit the proliferation and autophagy of OCSCs increased or decreased, respectively. Finally, analysis using a peripheral blood exosome database indicated that the contents of key factors (e.g.,ATF4,GPX4, andATG3) in peripheral blood exosomes from patients with ovarian cancer, were significantly higher than those of the healthy controls. Therefore, we hypothesized that anisomycin suppressed the expression of members of the glutathione metabolism and autophagy signal transduction pathways by downregulating the expression of ATF4. Moreover, anisomycin has the potential to induce human ovarian cancer stem cell ferroptosis. Overall, we confirmed that anisomycin has multiple targets and many mechanisms of action in inhibiting the activity of OCSCs.