The glycosylation reaction mediated by glycosyltransferases is not only a ubiquitous modification but also one of the structure diversity mechanisms for secondary metabolites synthesis. In this study, based on the pomegranate transcriptomic data, we isolated a UDP-glycosyltransferase gene (PgUGT) from peels of ‘Taishanhong’ pomegranate by RT-PCR. The basic physicochemical properties and phylogenetic relationships were analyzed by bioinformatics method. The gene expression profiles during fruit developmental stages were obtained by quantitative real-time PCR (qRT-PCR). By combination with total flavonoid content and total anthocyanin content analysis, the relationships between gene transcription levels and total flavonoid content and total anthocyanin content were discussed. Subsequently, the PgUGT gene was constructed on the prokaryotic expression vector and recombinant expression were performed. The results were as follows: (1) the PgUGT gene of pomegranate was successfully cloned (the access number in GenBank was MW414607). One open reading frame (ORF) of PgUGT gene was 1 557 bp, which encoded 518 amino acids. The molecular weight of protein was 55.9 kD, the isoelectric point was 6.55, the deduced protein was unstable hydrophilic one. PgUGT contained conserved PSPG motif, which belonged to GT-B glycosyltransferase superfamily. The phylogenetic trees analysis demonstrated that PgUGT was clustered into F group according to UGTs category of Arabidopsis thaliana, and was more closely related to that of grape and strawberry. (2) The qRT-PCR results demonstrated that the expression level of PgUGT exhibited a first-rise and then-fall pattern during fruit development, which was different from a gradual declining trend in total flavonoid content and gradual rising trend in total anthocyanin content. (3) The prokaryotic expression vector pCZN1-PgUGT was successfully constructed. The prokaryotic expression results showed that the recombinant plastid expressed in soluble form in E. coli expression systems. The size of fusion protein was approximately 58 kD as expected. The obtained results laid foundation for further revealing the roles and functions of PgUGT gene in flavonoid glycosylation reaction of pomegranate.