Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.