To investigate the proliferative effects and the synergistic mechanism of irinotecan combined with norcantharidin in human gastric cancer cell line BGC-823. BGC-823 cells were treated with CPT-11 30, 60, 90, 120, 150 μmol/L and NCTD 30, 60, 90, 120, 150 μmol/L alone or in combination with the fixed constant ratio (1∶1) and with the complete cross-over concentrations (as show above) for 24, 48 and 72 h, and the combination of CPT-11 and NCTD with a sequential schedule were combined for culturing BGC-823 cells for 24 h. Cell proliferation was investigated by MTT assay, and the combination effect was evaluated by Chou-Talalay method. Cell cycle and apoptosis in BGC-823 cells treated with CPT-11 60 μmol/L and NCTD 60 μmol/L alone or in combination (60∶60) μmol/L and combination with a sequential schedule for 24 h were determined by Flow cytometry. The expression of Pdcd4 and p53 in BGC-823 cells treated with CPT-11 30, 60 μmol/L and NCTD 30, 60 μmol/L alone or in combination (30∶30, 60∶60) μmol/L for 24 h was detected by Western blotting. Compared with treatment with CPT-11 and NCTD alone, the combination of them increased the proliferation inhibition, and the IC₅₀ were significantly decreased (P < 0.05). The IC₅₀ values of the combination for 24, 48 and 72 h were 2.83, 3.15, 2.19 fold and 2.66, 3.11, 2.45 fold respectively compared to BGC-823 cells treated with CPT-11 and NCTD alone. The results indicated synergistic effect. The sequence of CPT-11 followed by NCTD showed a stronger inhibition than the sequence of NCTD followed by CPT-11 (P < 0.05), and it was superior to co-administration (P < 0.05). For 24 h, CPT-11 60 μmol/L triggered both S and G2-M phase arrest (P < 0.01) in the BGC-823 cells, and NCTD 60 μmol/L induced cell cycle arrest at G2-M phases and apoptosis (P < 0.05) in the BGC-823 cells. The combination of CPT-11 and NCTD (60∶60) μmol/L increased the G2-M phase arrest. Compared with the sequence of NCTD 6 h first followed by CPT-11 and co-administration, the sequence of CPT-11 6 h first followed by NCTD increased the S phase arrest (P < 0.05) and apoptosis in the BGC-823 cells. CPT-11 30，60 μmol/L up-regulated the expression of Pdcd4 (P < 0.05) and down-regulated the expression of p53(P < 0.05) after 12 h; NCTD 30，60 μmol/L down-regulated the expression of Pdcd4 (P < 0.05) and up-regulated the expression of p53(P < 0.05) after 12 h; The combination (30∶30, 60∶60) μmol/L up-regulate the expression of Pdcd4 and p53(P < 0.05). The combination of CPT-11 and NCTD has a synergistic effect mainly due to the fact that it can induce the G2-M phase arrest, and up-regulate the expression of Pdcd4 and p53 as the tumor suppressor. The combination of CPT-11 and NCTD with a sequential schedule has some impacts on the growth of BGC-823 cells. The sequence of CPT-11 followed by NCTD shows a stronger inhibition than the sequence of NCTD followed by CPT-11 and co-administration, and it may be related to the increase of S phase arrest and apoptosis. Pdcd4 and p53 protein may have a negative regulation in BGC-823 cells.