Walnut (Juglans regia L.) is a major nut crop of the Juglandaceae family and is well-known for its high nutritional value, which is achieved by a rich array of polyphenolic compounds. Phenolics are considered beneficial to human health because of their antioxidant, antimutagenic, and free radical scavenging properties. However, the phenolic biosynthetic pathway in walnut remains poorly studied. In this study, we cloned a 5-enolpyruvylshikimate 3-phosphate synthase (JrEPSPS) gene from walnut, a key gene involved in the shikimate pathway that catalyzes the penultimate step of the shikimate pathway toward the biosynthesis of aromatic amino acids. Subsequent sequence analysis revealed that the JrEPSPS protein harbors an N-terminal helix-turn-helix-like motif, which is known to mediate EPSPS function by acting as a transcription factor and regulating the expression of genes in the phenylpropanoid pathway in poplar. Subcellar localization analysis suggested JrEPSPS was localized in chloroplasts. The transient overexpression of JrEPSPS in persimmon (Diospyros kaki Thunb.) leaves and fruit discs showed significantly increased phenolic accumulation by elevating the expression of phenolic biosynthetic pathway genes. These results provide novel insights into the roles of EPSPS involved in phenolic biosynthesis in plants.