We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g/g, dry weight, dw) and 0.17-0.25% artemisinin (g/g, dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g/g, dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that the overexpression of this AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis and show that its transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin.