Triptolide, a diterpene triepoxide derived from Trypterygium wilfordii, is documented to have antitumor activity in a broad range of solid tumors and leukemia. The mechanisms that are involved in triptolide-mediated apoptosis or growth inhibition in cancer cells are not fully understood. We identified a disintegrin and metalloproteinase 10 (ADAM10) as a novel molecular target of triptolide using affinity chromatography and mass spectrometry. The identification was confirmed by Western blot analysis using an anti-ADAM10 antibody. The expression of ADAM10 is enhanced in several tumors including leukemia and is involved in malignant cell growth and cancer progression. ADAM10 is a type 1 transmembrane glycoprotein that cleaves several plasma membrane proteins. We show that triptolide, at concentrations in the nM range, resulted in a significant decrease in ADAM10 expression followed by the appearance of ADAM10 cleaved product. Furthermore, triptolide reduced the viability of monocytic leukemic U937 cells. Triptolide treatment of MCF-7 breast cancer cells expressing ectopic ADAM10 or dominant negative ADAM10 (DN ADAM10) resulted in a decreased expression of ADAM10 with a concomitant increase in ADAM10 cleaved products. Moreover, siRNA-mediated knockdown of ADAM10 mRNA significantly affected the growth of MCF-7 cells. Interestingly, the combination of siRNA-mediated knockdown of ADAM10 mRNA expression and triptolide treatment lead to a further reduction in cell growth. Taken together, we provide evidence that ADAM10 is a novel target of triptolide, presenting a novel strategy to inhibit ADAM10 activity in tumorigenesis.