This study aimed to elucidate the mechanisms of diosgenin, a natural compound derived from traditional Chinese medicine, on the triple-negative breast cancer cell line MDA-MB-231, with a focus on its induction of cell cycle arrest and inhibition of cell migration. Multiple experimental approaches were employed, including RNA sequencing analysis, flow cytometry for cell cycle detection, wound healing assays to evaluate migration capacity, F-actin staining to observe cytoskeletal changes, transmission electron microscopy (TEM) to examine cellular morphology and filopodia alterations, as well as Western blot and immunofluorescence staining to detect the expression of related proteins. RNA sequencing revealed significant gene expression differences between the diosgenin-treated group and the control group, with KEGG and GO analyses showing enrichment of differentially expressed genes mainly in pathways related to cell cycle and migration. Flow cytometry demonstrated that diosgenin induced G₀/G₁ phase arrest in MDA-MB-231 cells in a dose-dependent manner. Further Western blot and immunofluorescence assays indicated that diosgenin regulated the cell cycle via the CDC25A-CDK2/4/6 axis. Wound healing assays showed that diosgenin significantly inhibited cell migration. F-actin staining indicated that diosgenin suppressed F-actin formation and disrupted its structure. TEM observations revealed a reduction in filopodia formation on the cell surface after diosgenin treatment. Moreover, Western blot and immunofluorescence results demonstrated that diosgenin inhibited migration by suppressing epithelial-mesenchymal transition (EMT) and the RhoA-ROCK1 signaling pathway. In conclusion, diosgenin induces G₀/G₁ phase arrest in MDA-MB-231 cells by regulating cell cycle-related proteins and significantly inhibits cell migration by suppressing F-actin formation, reducing filopodia on the cell surface, and inhibiting EMT and the RhoA-ROCK1 pathway.