<p>Gastric cancer is a leading cause of cancer-related mortality worldwide, emphasizing the need for novel therapeutic strategies. Actinobacteria are prolific producers of bioactive secondary metabolites with anticancer potential. In this study, we evaluated the antitumor activity of <i>Streptomyces</i> sp. M4 extract against the human gastric adenocarcinoma (AGS) cell line and explored its underlying mechanisms. The strain was cultured in starch casein broth, and metabolites were extracted using ethyl acetate. AGS cell were treated with the crude extract, and cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while apoptosis was analyzed by flow cytometry. Gene expression was measured using real-time PCR, and Caspase-3/7 activity was determined via colorimetric assays. The MTT assay revealed potent cytotoxicity with an IC₅₀ value of 26.15&#xa0;µg/mL at 24&#xa0;h. Annexin V–propidium iodide (PI) staining revealed that <i>Streptomyces</i> sp. M4 extract significantly induced apoptosis in AGS cells, as evidenced by a 79.35% increase in the apoptosis rate compared to that of the control group. Real-time PCR analysis demonstrated a 1.785-fold upregulation of <i>CASPASE-8</i>, whereas <i>CASPASE-9</i> expression decreased to 0.068-fold relative to control. Additionally, <i>BCL-2</i> was upregulated (4.752-fold), whereas <i>BAX</i> was downregulated (0.08-fold) in extract-treated cells. Caspase activity assays revealed a significant increase in Caspase-3/7 activity (0.761 mU/mL), indicating apoptosis induction. These findings suggest that <i>Streptomyces</i> sp. M4 extract exerts anticancer effects primarily through the extrinsic apoptotic pathway while modulating key survival and resistance mechanisms of cancer cells. This study underscores the potential of <i>Streptomyces</i> sp. M4 as a therapeutic agent in gastric cancer.</p>