<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Cervical cancer is the second-leading cause of cancer-related mortality in females. <jats:italic>Coix lacryma-jobi</jats:italic> L. var. ma-yuen (Rom.Caill.) Stapf ex Hook. f. is the most widely recognized medicinal herb for its remedial effects against inflammation, endocrine system dysfunctions, warts, chapped skin, rheumatism, and neuralgia and is also a nourishing food.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>To investigate the activity of <jats:italic>Coix lacryma-jobi</jats:italic> sprout extract (CLSE) on cell proliferation in human cervical cancer HeLa cells, we conducted a Cell Counting Kit-8 (CCK-8) assay. Flow-cytometric analysis and western blot analysis were performed to verify the effect of CLSE on the regulation of the cell cycle and apoptosis in HeLa cells.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We observed that CLSE significantly inhibited cell proliferation. Furthermore, CLSE dose-dependently promoted cell cycle arrest at the sub-G1/ S phase in HeLa cells, as detected by bromodeoxyuridine (BrdU) staining. The cell-cycle-arrest effects of CLSE in HeLa cells were associated with downregulation of cyclin D1 and cyclin-dependent kinases (CDKs) 2, 4, and 6. Moreover, CLSE induced apoptosis, as determined by flow-cytometric analysis and nuclear DNA fragmentation with Annexin V/propidium iodide (PI) and 4′6′-diamidino-2-phenylindole (DAPI) staining. Induction of apoptosis by CLSE was involved in inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and upregulation of the apoptotic proteins p53, cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved caspase-8. Finally, we observed that CLSE inactivated the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) pathways.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>CLSE causes cell cycle arrest and apoptotic cell death through inactivation of the PI3K/AKT pathway in HeLa cells, suggesting it is a viable therapeutic agent for cervical cancer owing to its anticancer effects.</jats:p> </jats:sec>