Although phytohormones are known to be important signal molecules involved in wood formation, their roles are still largely unclear. Here, Populus simonii × P. nigra seedlings were treated with different concentrations of exogenous phytohormones, indole-3-acetic acid (IAA), gibberellin (GA₃), and brassinosteroid (BR), and the effects of phytohormones on growth were investigated. Next, 27 genes with known roles in wood formation were selected for qPCR analysis to determine tissue-specificity and timing of responses to phytohormone treatments. Compared to the control, most IAA, GA₃, and BR concentrations significantly increased seedling height. Meanwhile, IAA induced significant seedling stem diameter and cellulose content increases that peaked at 3 and 30 mg·L⁻¹, respectively. Significant increase in cellulose content was also observed in seedlings treated with 100 mg·L⁻¹ GA₃. Neither stem diameter nor cellulose content of seedlings were affected by BR treatment significantly, although slight effects were observed. Anatomical measurements demonstrated improved xylem, but not phloem, development in IAA- and BR-treated seedlings. Most gene expression patterns induced by IAA, GA₃, and BR differed among tissues. Many IAA response genes were also regulated by GA₃, while BR-induced transcription was weaker and slower in Populus than for IAA and GA₃. These results reveal the roles played by phytohormones in plant growth and lay the foundation for exploring molecular regulatory mechanisms of wood formation in Populus.