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Tree peony (<i>Paeonia ostii</i>) is an ornamental flowering plant that is generally recalcitrant to establishment of a mature somatic embryo regeneration system in vitro. Glucose-6-phosphate translocator (GPT) plays an important regulatory role in embryogenesis of plants. In this study, <i>PoGPT1</i> was cloned, and a bioinformatic analysis and functional verification of the gene were performed. The results showed that <i>PoGPT1</i> encoded a polypeptide of 392 amino acids, which was a basic non-secreted hydrophobic transmembrane protein, and was mainly localized in the plastids. <i>PoGPT1</i> was highly expressed in tree peony leaves, and its transcript abundance increased with the progression of zygotic embryo development. Overexpression of <i>PoGPT1</i> caused up-regulation of leafy cotyledon 1 (<i>PoLEC1</i>), somatic embryogenesis receptor-like kinase (<i>PoSERK</i>), and agamous-like15 (<i>PoAGL15</i>) in tree peony callus. In addition, <i>PoGPT1</i> overexpression promoted the increase in indole-3-acetic acid (IAA), 5-deoxystrigol (5DS), and brassinolide (BL) contents, especially of IAA, but reduced the contents of abscisic acid (ABA), 6-benzyladenosine (BARP), and 1-aminocyclopropanecarboxylic acid (ACC). The present research showed that <i>PoGPT1</i> synergistically regulated the contents of endogenous hormones and expression levels of embryogenesis-related genes to promote the embryonic development of tree peony. The results provide theoretical and technical support for the establishment of a tree peony embryogenic callus formation and subsequent research on somatic embryogenesis.