Abstract: To study the effect of brassinolide (BR) on tobacco stem growth and elongation, the tobacco seedlings were treated with 2,4-epibrassinolide (EBR) of two different concentrations 0.5×10^-7 mol/L (T1), 0.5×10^-5 mol/L (T2) and distilled water was used as control (CK). Plant height, internode distance, and stem anatomical structure were analyzed. Genes related to cell division and cell size regulation, and genes related to biosynthesis of BR, auxin (IAA), and gibberellin (GA) were also analyzed. The results showed that the application of EBR increased the seedling pitch and plant height, the promotion effect on stem growth was enhanced with increment of EBR concentrations. Stem anatomical study revealed an increase in the number of parenchyma cells and a decrease in its area after EBR treatment. EBR upregulated cell cycle-related gene NtCYCD3 while downregulated NtARF6 and NtARF16 genes that are related to cell size. Similarly, BR signaling receptor genes NtBRI1 and NtBIN2 were upregulated while the BR transcription factor NtBES1T was downregulated in response to EBR application. The expression of key biosynthesis genes NtDWF4, NtYUCCA8, and NtGA3ox-2 of BR, IAA, and GA, respectively, were all upregulated. The results showed that the application of EBR promoted the expression of endogenous BR, IAA and GA biosynthesis genes, and helped in the growth and elongation of tobacco seedling stem by promoting the division of internode cells and inhibiting cell expansion.