Background: Marsdenia tenacissima extract (MTE) is a traditional Chinese medicine that can be effectively used against various cancers. However, to the best of our knowledge, its role in ovarian cancer is not known. This study investigated the effects of MTE on human ovarian cancer SKOV3 cells. Material/Methods: The viability and cell cycle of SKOV3 cells were assessed using the cell counting kit-8 (CCK-8) and propidium Iodide (PI) staining kit, respectively. Cell apoptosis and mitochondrial membrane potential (MMP) were detected by flow cytometry. The expression levels of proliferation-related and apoptosis-related factors were tested by quantitative real-time PCR (qRT-PCR) and Western blot assays, respectively. Results: We found that MTE markedly reduced the viability of SKOV3 cells in dose-dependent and time-dependent manners. MTE induced cell cycle arrest by downregulating the levels of cyclin D1 and cyclin B1. MTE (10, 20, and 40 mg/mL) markedly increased apoptosis rates (2.77 +/- 0.6%, 4.95 +/- 0.97%, and 12.16 +/- 0.69%, respectively), and enhanced the loss of MMP. MTE obviously downregulated the expression of B cell lymphoma-2 (Bcl-2) and up-regulated the expression levels of fibroblast-associated (Fas), Fas ligand (FasL), cleaved cysteinyl aspartate-specific proteinas-3 (caspase-3), and Bcl-2-associated X protein (Bax) compared to the control group. In addition, the expressions of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated protein kinase B (p-AKT), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) were decreased by MTE. Conclusions: MTE inhibited proliferation and induced apoptosis of SKOV3 cells. The depression of the PI3K/AKT/mTOR pathway may augment the protective effect of MTE. Thus, MTE might be expected to be a new drug for curing ovarian cancer.