Abstract: To identify key transcription factors regulating proanthocyanidin biosynthesis in the succulent plant Aeonium arboreum ‘Halloween’, the AaMYB5 gene was cloned based on transcriptome data, and sequence analysis, promoter element prediction, transcriptional activity assay, and functional verification were performed. The results showed that the open reading frame of AaMYB5 is 984 bp in length, encoding a protein of 327 amino acids, and contains two exons and one intron. The AaMYB5 protein is basic, unstable, and hydrophilic, with a secondary structure predominantly composed of random coils. Protein sequence alignment and phylogenetic analysis indicated that AaMYB5 belongs to the R2R3-MYB transcription factor family, possesses conserved motifs characteristic of MYB proteins involved in the regulation of proanthocyanidin biosynthesis, shares 51.2% protein sequence similarity with the grape proanthocyanidin regulator VvMYBPA1, and clusters with MYB known to positively regulate proanthocyanidin biosynthesis. Promoter cis-element analysis revealed that the 2000 bp upstream region of AaMYB5 contains, in addition to core promoter elements, ten light-responsive elements as well as elements responsive to abscisic acid, drought, and multiple hormones, suggesting that its expression may be regulated by light and abiotic stress signals. Yeast transcriptional activity assays confirmed that AaMYB5 possesses transcriptional activation activity. Furthermore, an AaMYB5 overexpression vector was constructed and transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. The results showed that proanthocyanidin content was significantly increased in tobacco leaves overexpressing AaMYB5 (P<0.05) , and the expression levels of proanthocyanidin biosynthesis-related structural genes, including NtPAL, NtCHS, NtDFR, NtLAR, and NtANR, were significantly upregulated (P<0.05) . This study confirms that AaMYB5 is a key transcription factor promoting proanthocyanidin biosynthesis in Aeonium arboreum ‘Halloween’, laying a foundation for further elucidating the molecular mechanism by which AaMYB5 regulates proanthocyanidin biosynthesis in succulents and for its potential application in the quality improvement of ornamental plants.