An inverse metabolic engineering approach has been applied for an improved carotenoid production in Escherichia coli. Elementary Mode Analysis, a stoichiometry metabolic model, was used for a rational design of a carotenoid over-producing strain by identifying target genes knockout for elimination of inefficient carotenoid biosynthesis pathway. Therefore, on the basis of the model, the designed strain would produce carotenoid product in an efficient manner. To validate the model, we have constructed the designed strain CRT028 and tested its performance on carotenoid synthesis. This multiple-genes knockout strain has experimentally revealed up to 5 times improvement on carotenoid product yield as compared with the wildtype strain under identical growth condition. The consistency between modeling and experimental result has proven Elementary Mode Analysis as a tool for guiding the rational direction in the cell's metabolic engineer.