Objective To investigate the inhibitory effect of luteolin against proliferation of gastric cancer cell line MGC803 and the related mechanism. Methods The inhibitory effect of luteolin against MGC803 cell proliferation was evaluated by MTT assay and clone forming assay. The cell cycle changes were analyzed by the flow cytometry after staining propidium iodide (PI). Annexin Ⅴ-PI double staining was used to determine the apoptosis ratio of MGC803 cells. Bcl-2 family proteins, Caspase proteins, autophagy-associated and cell cycle-associated proteins were assessed by Western blotting analysis. Results MTT assay results showed that the IC₅₀ of luteolin against MGC803 cells for 24 h, 48 h and 72 h treatment were 127.37 μmol/L, 76.12 μmol/L, and 16.84 μmol/L, respectively. Luteolin treatment also inhibited cell colony formation of MGC803 cells. PI single staining flow cytometry found that cells were arrested in G₂ phase with the increase of luteolin concentration. Western blotting results showed that, with the increase of luteolin concentration, CDK4, CDK6 and CyclinE2 protein expression was decreased; CyclinD1, CyclinD3 and p-Wee1 protein kept unchanged; and CyclinA, CyclinB, Myt1 and p-cdc2 (Tyr15) protein expression was decreased. Flow cytometry Annexin Ⅴ-FITC/PI revealed increase of early and late apoptosis with the increase of luteolin concentration. Western blotting analysis showed that Mcl-1, Bcl-x_L, Caspase-3, Caspase-8 and Caspase-9 protein expression was decreased with the increase of luteolin concentration, with slightly changed Bcl-2 expression and increased Bax expression, which led to decreased Bcl-2/Bax, Mcl-1/Bax and Bcl-x_L/Bax ratios. At the same time, Caspase-3 and PARP also appeared in the strip cutting. The expression of P62 was decreased and expression of Beclin-1 and LC3 Ⅱ was increased. Conclusion Luteolin can arrest gastric cancer MGC803 cells at G₂ phase, inducing autophagy and inhibiting cell growth. Luteolin can also induce apoptosis of MGC803 cells via the mitochondrial pathway.