Abstract: MYB transcription factors play important roles in plant growth and development, and are involved in biological and abiotic stress responses. In order to study the gene functions of MYB transcription factors in tobacco, we cloned a MYB transcription factor gene from tobacco K326 by homologous cloning. The bioinformatics analysis of gene sequences and gene coded amino acids showed that this gene had a typical MYB domain and was named as NtMYB59. The total length of NtMYB59 CDS was 666 bp, encoding 221 amino acids. The predicted molecular weight of NtMYB59 protein was 26.26 kDa, and the predicted isoelectric point was 8.33. Secondary structural analysis revealed that NtMYB59 contained a unique helix-coil-helix domain of MYB protein, and its tertiary structural prediction suggested that it was consistent with the structural characteristics of the MYB gene family. Real-time quantitative PCR was used to analyze the expression of NtMYB59 gene in different tissues of tobacco, and it was found that the expression was higher in leaves, lateral roots, fibrous roots and flowers, and lower in stems, stem nodes and axillary buds. The chlorogenic acid content (mass fraction) in transgenic tobacco leaves with NtMYB59 gene was significantly increased compared with K326, indicating that NtMYB59 transcription factor gene played a role in regulating the chlorogenic acid content in tobacco.