Pancreatic cancer is a devastating disease characterized by low sensitivity to conventional chemotherapeutic treatment that has a poor prognosis. Therefore, novel effective chemotherapeutic regimens need to be developed. In this study, we analyzed the combined cytotoxic effect of triptolide and hydroxycamptothecin (HCPT) on pancreatic cancer cell line PANC-1 by using 3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and fluorescence- activated cell sorting (FACS) assays. Our results showed that the sensitivity of a combined therapy using triptolide and HCPT was higher than that of triptolide or HCPT alone and that activation of caspase-9/caspase-3 and inhibition of nuclear factor-kappaB (NF-κB) signaling pathway may contribute to the synergistic cytotoxic effect of this combination therapy. Therefore, our observations provided evidence supporting the clinical applications of the combination chemotherapy using triptolide and HCPT for treating pancreatic cancer.