Gastric cancer cells are usually insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In the present study, in MGC803 cells treated with 100 ng/ml TRAIL for 24 h, the inhibition rate of cell proliferation was 9.76±2.39% and the rate of cell apoptosis was only 4.37 ± 1.45%. Treatment with epirubicin (1.18 µg/ml, IC50 dose for 24 h) and TRAIL (100 ng/ml for 24 h) led to a marked increase in the inhibition rate of cell proliferation and apoptosis compared to treatment with epirubicin or TRAIL alone (P<0.05). Moreover, even more notable cleavage of caspase-3 and 8 was detected with the combination of epirubicin and TRAIL. TRAIL (100 ng/ml) induced only light lipid raft aggregation and DR4 and DR5 clustering. Epirubicin significantly promoted lipid raft DR4 and DR5 aggregation, as well as the localization of DR4 and DR5 in the lipid rafts. Similar results were detected with the combination of epirubicin and TRAIL. Pretreatment with 50 µg/ml nystatin, a cholesterol-sequestering agent, partially prevented epirubicin-induced lipid raft aggregation and DR4 and DR5 clustering. Pretreatment with nystatin did not markedly inhibit epirubicin-induced apoptosis, while nystatin partially suppressed epirubicin and TRAIL-induced apoptosis (P<0.05). Our data demonstrate that epirubicin enhanced TRAIL-induced apoptosis in gastric cancer MGC803 cells, at least partially, through death receptor redistribution in the lipid rafts.