Protandim is an antioxidant supplement that consists of five ingredients, namely, ashwagandha, bacopa extract, green tea extract, silymarin, and curcumin, each with known therapeutic properties. Protandim was formulated with the objective of combining multiple phytochemicals at low nontoxic doses to gain synergy among them. A recent clinical study demonstrated the in vivo antioxidant effects of Protandim (S.K. Nelson et al., 2006, Free Radic. Biol. Med. 40, 341-347). The objective of the present study was to determine if the components of Protandim induce heme oxygenase-1 (HO-1) in a synergistic manner in cultured MIN6 cells, a mouse beta-cell line, and in SK-N-MC cells, a human neuroblastoma cell line. When the components of Protandim were tested alone at low doses, curcumin showed minimal induction, whereas the others were unable to induce the HO-1 promoter, assayed by transient transfection. All components together, however, produced a strongly synergistic induction of around three- to ninefold in a dose-dependent manner, greatly exceeding the sum of the parts. Similar findings were obtained for the expression of HO-1 at the mRNA and protein levels. Protandim-mediated HO-1 induction involved the presence of ARE sites in the HO-1 promoter and nuclear translocalization of the transcription factor Nrf2, which binds to ARE sites. The involvement of multiple signaling pathways, including PI3-kinase/Akt, p38MAPK, and PKCdelta, in HO-1 induction seems to be the probable mechanism of synergy between the components of Protandim. There were significant increases in the levels of total glutathione in Protandim-treated cells. These findings suggest that the use of a combination of phytochemicals may be an efficient method for the induction of antioxidant enzymes.