Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs, because the pathway is essential in microorganisms, plants, and parasites but absent from mammals. SK catalyzes the reaction of phosphoryl transfer from ATP to shikimic acid (SA). Since 2002, a total of 11 SK structures have been reported, but none contains either the two substrate (SA and ATP) or the two product (SA-phosphate and ADP) molecules. Here, we present three crystal structures of SK from Mycobacterium tuberculosis (MtSK), including apo-MtSK, a binary complex MtSK x SA, and the ternary complex of MtSK with SA and an ATP analogue, AMPPCP. The structures of apo-MtSK and MtSK x AMPPCP x SA make it possible to elucidate the conformational changes of MtSK upon the binding of both substrates; the structure of MtSK x AMPPCP x SA reveals interactions between the protein and gamma-phosphate which indicate dynamic roles of catalytic residues Lys15 and Arg117.