〔Abstract〕 Objective To investigate the effect of disthiolam (DSF) on the malignant degree of human ovarian cancer cells and its sensitivity to chemotherapy drugs and related mechanisms. Methods Ovarian cancer cell lines SK-OV-3 and A2780 were treated with different concentrations of DSF (0.1 μmol·L-1, 1 μmol·L-1, 5 μmol·L-1), and 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyl-trazolium bromide (MTT) assay was used to determine cell viability. The effective concentration of DSF (5 μmol·L-1) was selected to treat the cells, and the characteristics of stem cells were detected by soft agarose formation assay and microsphere formation assay, cell migration was detected by Transwell assay, and messenger ribonucleic acid (mRNA) expression of Wnt target gene was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The transcriptional activity of Wnt pathway was detected by dual luciferase assay, and the activation of PI3K/AKT/mTOR and RAF/MEK/ERK was detected by Western blot assay. SK-OV-3 and A2780 cells were treated with DSF (5 μmol·L-1) and cisplatin, and the effects of DSF on the sensitivity of Cisplatin were detected by MTT assay, soft agron clonogenesis assay and microsphere formation assay. Results Compared with the control group, DSF treatment significantly reduced the proliferation ability of ovarian cancer cells (P < 0.01), decreased the cloning ability of cancer cells in soft agarose (P < 0.01), inhibited the formation of microspheres in suspension culture (P < 0.01) and migration ability of ovarian cancer cells (P < 0.01), so as to reduce the malignancy of ovarian cancer cells. DSF combined with cisplatin significantly increased the sensitivity of ovarian cancer cells to cisplatin (P < 0.01). Studies on mechanism showed that DSF treatment significantly reduced the activity of Wnt signaling pathway in ovarian cancer cells, and significantly inhibited the activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in ovarian cancer cells. Conclusion DSF treatment can significantly reduce the degree of malignancy of ovarian cancer cells and enhance their sensitivity to traditional chemotherapy drug cisplatin. The anticancer effect of DSF may be realized by inhibiting the activity of cancer-promoting signaling pathways such as Wnt, RAF/MEK/ERK and PI3K/AKT/mTOR.