The inhibitory effect of matrine (MA) was studied in combination with irinotecan (CPT-11) on proliferation of human colon carcinoma cell line HT29. We also explored the mechanism of cell apoptosis induction in HT29. HT29 cells were treated with different concentrations of MA and CPT-11 alone and in combination. The growth inhibition in HT29 cells was evaluated using MTT assay. Apoptosis was detected using AV-PI double staining flow cytometry. Transmission electron microscopy was used to detect structural changes in cells. Topoisomerase (TOPO) I, Bax and Caspase-3 expression levels were evaluated using western blot analysis. MA and CPT-11 alone and in combination, inhibited the proliferation of HT29 cells, whereas the combination treatment exhibited higher inhibitory effect (P<0.01). This suggests the existence of synergistic cytotoxicity. Compared with each treatment alone, the combination treatment caused more significant damage to cell structure, and caused a significantly higher apoptosis rate (P<0.01). Additionally, the combination treatment increased TOPO I, Bax and Caspase-3 expression levels (P<0.01). In conclusion, MA in combination with CPT-11 synergistically inhibited HT29 cell proliferation and induced apoptosis in these cells. The mechanism may be related to upregulation of the TOPO I, Bax and Caspase-3 protein expression.