20(S)-Ginsenoside Rg3 (GRg3) has various bioactivities including anti-cancer effects and inhibition of autophagy. However, no reports have investigated the appearance of autophagy or the connection between autophagy and apoptosis in HeLa cells treated with 20(S)-GRg3. Cell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis and the cell cycle were analyzed by Hoechst 33342 staining and flow cytometry. Apoptotic pathways were examined by ROS (reactive oxygen species) determination and rhodamine 123 assays. Western blot analysis was used to determine changes in protein levels. Autophagy induction was monitored by acidic vesicular organelle staining and EGFP-LC3 transfection. 20(S)-GRg3 inhibited autophagy of cells in a starved state, making it impossible for cells to maintain a steady state through autophagy, and then induced apoptosis. 20(S)-GRg3 blocked the late stage of autophagy (fusion of lysosomes and degradation of autophagic lysosomes), including a decrease in acidic vesicular organelle fluorescence, increased LC3 I–II conversion, accumulation of EGFP-LC3 fluorescence, GFP-mRFP-LC3 red-green fluorescence ratio, degradation of the substrate p62, and loss of the balance between autophagy and apoptosis, which induced apoptosis. ROS increased, the mitochondrial membrane potential decreased, apoptotic inducer AIF was released from mitochondria, and nuclear transfer occurred, triggering a series of subsequent apoptotic events. Autophagy inducer rapamycin inhibited the apoptosis induced by 20(S)-GRg3, whereas autophagy inhibitor BA1 promoted apoptosis induced by 20(S)-GRg3. Therefore, 20(S)-GRg3 promoted HeLa cell apoptosis by regulating autophagy. In the autophagic state, 20(S)-GRg3 can be used as a novel autophagy inhibitor in synergy with tumor-blocking therapies such as chemotherapy, which supports its application in the medical field.