Ovarian cancer is a deadly gynecological malignancy often diagnosed at an advanced stage, with few treatment options available. Polyphyllin I (PPI), the primary active ingredient of Paris polyphylla, has shown excellent anti-cancer activity against various cancers. However, studies on PPI use and ovarian cancer are limited, and the potential mechanisms remain unclear. This study explored the effects of anti-ovarian cancer and the potential mechanisms of action of PPI. Methods such as MTT, colony formation, three-dimensional spheroid formation, flow cytometry, and western blotting were employed to assess the anti-proliferative effects of PPI on different ovarian cancer cell lines. RNA sequencing was used to discover potential mechanisms by evaluating the effects of PPI on transcriptional signatures and identifying differences in gene expression patterns. The results indicate that PPI demonstrated dose- and time-dependent anti-proliferative effects on ovarian cancer cell lines. PPI also up-regulated 1518 genes and down-regulated 800 genes. The genes showed enrichment in the "apoptosis" gene sets, while exhibiting negative enrichment in the "cell cycle" and "Myc signaling pathway" gene sets. PPI caused cell cycle arrest at the G0/G1 phase by increasing the expression of p21 and p27 and inhibiting the activity of the CDK2/cyclin E complex. PPI triggered cell apoptosis, which was associated with elevated cellular reactive oxygen species and mitochondrial membrane potential depolarization. Finally, PPI reduced c-Myc protein levels and affected mRNA levels of c-Myc pathway-related genes. PPI effectively suppressed ovarian cancer cell proliferation, indicating its potential as a treatment option for ovarian cancer.