Transcription factor HY5 (ELONGATED HYPOCOTYL5) played an important role in anthocyanin biosynthesis. In this study, we firstly cloned PdHY5 gene from the Populus deltoides ‘Quanhong’ and analyzed it by bioinformatics methods, we then transformed PdHY5 gene into tobacco by Agrobacteriummediated leaf disc method for functional analysis. The results showed that: (1) the open reading frame (ORF) of PdHY5 was 510 bp and encoded 169 amino acids. Comparison of amino acid sequences and phylogenetic analysis indicated that PdHY5 contained a conserved domain of bZIP family and had a closest relationship with PtHY5. (2) An overexpression vector pNP130235SPdHY5 was successfully constructed, and 4 tobacco transgenic lines (S1-S4) were obtained by hygromycin screening. (3) qRTPCR results showed that the expression of PdHY5 gene in the 4 overexpression lines was significantly higher than that of WT (wild type), with the highest expression of S4 and the least of S1. Whats more, the expression levels of several key genes participated in anthocyanin biosynthesis, such as CHS, F3H, and FLS in overexpression lines were significantly upregulated compared with WT. (4) The relative content of anthocyanin was significantly higher in transgenic tobacco lines S2, S3 and S4 compared with WT, increasing by 128.23%, 97.36% and 134.20%, respectively. This study indicated that overexpression of PdHY5 gene regulated the expression of structural genes in the anthocyanin biosynthesis pathway of tobacco itself, thereby promoting the accumulation of anthocyanins.