(R)-Shikonin/(S)-Alkannin and their derivatives (collectively denoted as shikonin) are abundantly distributed in the roots of Boraginaceae plants. Acetylshikonin, as a crucial derivative of (R)-shikonin, possesses substantial research significance. Previous investigations have shown that LeSAT2, specifically catalyzes the acylation of (R)-shikonin to yield various acylated (R)-shikonin derivatives, including acetylshikonin. Additionally, electrophoretic mobility shift assays (EMSA) indicated that LeWRKY24 transcription factors may participate in the transcriptional regulation of LeSAT2. In this study, the interaction between LeWRKY24 and LeSAT2 was further verified using yeast one-hybrid and dual-luciferase assays. The LeWRKY24 overexpression vector was constructed and introduced into Echium plantagineum to induce transgenic hairy roots. High-performance liquid chromatography (HPLC) analysis showed that in LeWRKY24-overexpressing hairy root lines, the total pigment content was significantly increased, and in particular, the content of acetylshikonin was specifically upregulated by as much as 200‐fold. Transcriptome data revealed that genes associated with shikonin biosynthesis in the overexpression lines were significantly upregulated, activating the metabolic pathway, which explains the molecular mechanism underlying this regulatory effect. Our results also confirm that LeWRKY24 possesses cross-species regulatory universality in shikonin biosynthesis, laying a foundation for its widespread application in the metabolic engineering of Boraginaceae plants.