Background: The medicinal phytochemical oleandrin (Ole) is obtained from the Nerium oleander plant. The exact relationship between Ole-induced apoptosis and autophagy in gastric cancer (GC) is unclear despite the fact that it has outstanding anti-tumor capabilities. This research aimed to demonstrate how autophagy and Ole-induced apoptosis interact in GC. Methods: The Cell Counting Kit (CCK)-8 assay and colony formation assays were employed to evaluate cell proliferation. Cellular apoptosis was evaluated with Calcein/Propidium Iodide (PI) assays and flow cytometry. Confocal and electron microscopes were employed to examine the morphology of autophagy. Protein concentrations were assessed by western blotting. Luciferase-positive HGC-27 cells were administered subcutaneously to Balb/c nude mice to evaluate Ole’s anti-tumor activity. Immunohistochemistry assessed Ki67 expression and H&E staining in tumor tissue. Results: Ole causes GC cells to undergo intracellular apoptosis and autophagy at low nanomolar doses, halting the cell cycle at the G0/G1 phase. Whereas 3-methyladenine (3-MA), the inhibitor of autophagy, counteracts the apoptosis generated by Ole in vitro and in vivo. Conclusions: Ole may trigger apoptosis through the activation of autophagy in GC. It offers a secure and efficacious candidate drug for the treatment of tumors in the digestive system.