We previously reported that the transient and stable expression of IbMYB1a produced anthocyanin pigmentation in tobacco leaves and transgenic Arabidopsis plants, respectively. To further determine the effects of different promoters on the expression of IbMYB1a and anthocyanin production, we generated and characterized stably transformed tobacco (Nicotiana tabacum SR1) plants expressing IbMYB1a under the control of three different promoters. We compared the differences in anthocyanin accumulation patterns and phenotypic features of the leaves of these transgenic tobacco plants during growth. Expression of IbMYB1a under the control of these three different promoters led to a remarkable variation in anthocyanin pigmentation in tobacco leaves. The anthocyanin contents of the leaves of the SPO-IbMYB1a-OX (SPO-M) line were higher than those of the SWPA2-IbMYB1a-OX (SPA-M) and 35S-IbMYB1a-OX (35S-M) lines. High levels of anthocyanin pigments negatively affected plant growth in the SPO-M lines, resulting delayed growth and, occasionally, a stunted phenotype. Furthermore, HPLC analysis revealed that transcriptional regulation of IbMYB1a led to the production of cyanidin-based anthocyanins in the tobacco plants. In addition, RT-PCR analysis revealed that IbMYB1a expression induced the up-regulation of several structural genes in the anthocyanin biosynthetic pathway, including DFR and ANS. Differential expression levels of IbMYB1a under the control of different promoters were highly correlated with the expression levels of the structural genes, thereby affecting anthocyanin production levels. These results indicate that IbMYB1a positively controls the expression of multiple anthocyanin biosynthetic genes and anthocyanin accumulation in heterologous tobacco plants.