Background: Flavonoids constitute one of the best-characterized groups of plant secondary metabolites with enormous pharmaceutical potential. A flavone type of plant flavonoid, cirsilineol, has been reported to exhibit proapoptotic effects against malignant human cells. Objectives: The present study was designed to investigate the antiproliferative effects of cirsilineol against human gastric cancer cells. Materials and Methods: Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. Apoptosis was detected by acridine orange/ethidium bromide (AO/EB) and annexin V/propidium iodide (PI) assay. Protein expression was examined by western blotting analysis. Results: The results showed cirsilineol inhibits the proliferation of human gastric cancer cells. The IC50 of cirsilineol against human gastric cancer cells (BGC-823, SGC-7901, and MGC-803) ranged from 8 to 10 μM. Nonetheless, cirsilineol exhibited comparatively lower antiproliferative effects against normal GES-1 cells. The IC50 of cirsilineol against normal GES-1 cells was found to be 120 μM. Colony formation assay showed that cirsilineol suppressed the colony formation of BGC-823 and MGC-803 cells in a dose-dependent manner. Acridine orange and ethidium bromide (AO/EB) staining showed that cirsilineol induced apoptosis in BGC-823 and MGC-803 cells. The percentage of apoptosis increased from 7.4% in control to 40.5% in BGC-823 cells and from 6.56% in control to 33.53% in MGC-803 cells at 8 μM cirsilineol. Western blotting showed cirsilineol caused an increase in Bax and cleaved caspase-3 and a decrease in Bcl-2 expression in both BGC-823 and MGC-803 cells. Conclusion: Together, the results are indicative of the proapoptotic and antitumor potential of cirsilineol against gastric cancer cells, suggestive of its possible therapeutic significance in future.