Artemisinin is an endoperoxide sesquiterpenelactone isolated from the aerial parts of Artemisia annua L.,and is presently the most potent anti-malarial drug. Owingto the low yield of artemisinin from A. annua as well as thewidespread application of artemisinin-based combinationtherapy recommended by the World Health Organization,the global demand for artemisinin is substantially increasingand is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is toincrease the content of artemisinin in A. annua. In thisstudy, three key genes in the artemisinin biosynthesispathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochromeP450 reductase, were over-expressed in A. annuathrough Agrobacterium-mediated transformation. Thetransgenic lines were confirmed by Southern blotting andthe over-expressions of the genes were demonstrated byreal-time PCR assays. The HPLC analysis showed that theartemisinin contents in transgenic lines were increasedsignificantly, with the highest one found to be 3.6-foldhigher (2.9 mg/g FW) than that of the control. Theseresults demonstrate that multigene engineering is aneffective way to enhance artemisinin content in A. annua.