The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of Tyr with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan (Trp) and phenylalanine (Phe) supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, BiFC studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol which prevents it ability to function in the chloroplast with elevated Tyr.