Based on the wingless MMTV integration site family (Wnt)/β-catenin (β-catenin) pathway, the effect of Marsdenia tenacissima C21 steroid saponin (MTC21) on cisplatin resistance in ovarian cancer cells and related molecular mechanisms were analyzed. SKOV3/DDP cell line with cisplatin-resistant ovarian cancer was selected. The proliferation of SKOV3/DDP cells under different concentrations of DDP (0, 1, 2, 4, 8, 16 μg/mL) was detected using CCK-8. The control group (Con), cisplatin group (DDP; 2 μg/mL) and the MTC21 in Low-dose group (L-MTC21), MTC21 in Medium-dose group (M-MTC21), and MTC21 in High-dose group (H-MTC21) (2 μg/mL DDP and 0.05, 0.2, 0.8 mg/mL MTC21 interventions) were set up. Flow cytometry was used to detect the apoptosis of each group, and the mRNA and protein expressions of Wnt and β-catenin were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The dose-response curve showed that the 50% concentration of inhibition (IC₅₀) of DDP against SKOV3/DDP cells in the Con group was 10.9 μg/mL. The IC₅₀ of the L-MTC21, M-MTC21 and H-MTC21 groups were 9.1, 8.3 and 6.5 μg/mL, respectively. With the increase of the concentration of MTC21, the IC₅₀ value of DDP decreased on cells, and the IC₅₀ value of H-MTC21 group was the lowest. Compared with the Con group, the apoptosis rate of the DDP group, L-MTC21, M-MTC21 and H-MTC21 groups increased (P<0.05), and the mRNA and protein expressions of Wnt and β-catenin decreased (P<0.05). Compared with the DDP group, the apoptosis rate of the L-MTC21, M-M-MTC21 and H-MTC21 groups increased (P<0.05), and the mRNA and protein expressions of Wnt and β-catenin decreased (P<0.05). Compared with the L-MTC21 group, the apoptosis rate of the M-MTC21 group and H-MTC21 group increased (P<0.05), and the mRNA and protein expressions of Wnt and β-catenin decreased (P<0.05). Compared with the M-MTC21 group, the apoptosis rate of the H-MTC21 group was increased (P<0.05), and the mRNA and protein expressions of Wnt and β-catenin were decreased (P<0.05). In conclusion, MTC21 can increase the sensitivity of ovarian cancer SKOV3/DDP cells to DDP chemotherapy, and its mechanism of action may be related to the inhibition of Wnt/β-catenin pathway.