To investigate the inhibitory effect of ginsenosides Rg5 on cancer cells, the underlying mechanisms, and its safety profile, a CCK-8 assay was conducted to screen for sensitive cancer cell lines. Rg5 mechanisms and safety were evaluated using various assays. Flow cytometry was applied to evaluate Rg5 apoptotic and cell cycle modulating effects in sensitive cancer cells. Rg5’s inhibitory effects on cancer cell migration and invasion were examined using scratch and Transwell assays. Western blot analysis was performed to evaluate Rg5’s effect on the expression of certain cell proteins. Complexes of Rg5 with MMP-2 or MMP-9 were simulated using Auto Dock. The safety of Rg5 was evaluated in vivo in animal experiments. Among Hela, MCF-7, HCT116, HepG2, A549, SiHa, and CaSki cells, Hela cells were the most sensitive to Rg5, with a 72 h-IC50 value of 53.02 μmol•L-1. Rg5 could induce Hela cell apoptosis, arrest Hela cells at G2/M phase, and inhibit HeLa cell migration and invasion. Western Blot analyses demonstrated that Rg5 could activate the mitochondrial apoptosis pathway and reduce the expression of MMP-2, MMP-9, E-cadherin, and cancer cell stemness factors. Auto Dock simulation indicated that Rg5 could bind within the hydrophobic pockets of MMP-2 and MMP-9. In vivo safety experiments showed that Rg5 caused no significant changes in liver and kidney function, routine blood parameters, or major organs in mice. As a natural product, Rg5 can induce apoptosis by activating the apoptotic pathway and can suppress Hela cell invasion by downregulating the expression of cancer stemness related factors and associated proteins. The results provide theoretical support for further development and utilization of Rg5 in functional food and health products.