In the present study, an attempt was made to induce rooting from single-node cuttings of hybrid aspen (Populus tremula L. × P. tremuloides Michx.) with different concentrations of Indole-3-acetic acid (IAA), Indole-3-Butytric acid (IBA) and 1-Naphthylacetic acid (NAA). Among the three auxins used, 0.54 mM NAA showed more effective induction on rooting as compared to IAA and IBA at the whole level. Thereafter, 0.54 mM NAA was used further for the anatomical and biochemical investigation. The results showed that it took 12 days from the differentiation of primordium to the appearance of young adventitious roots under NAA application. It was found that endogenous IAA, Zeatin riboside (ZR) and Gibberellic Acid (GA3) levels increased, but Abscisic acid (ABA) decreased in cuttings with NAA treatment. In contrast to the endogenous IAA level, NAA resulted in a decrease in IAA-oxidase (IAAO) activity. Similarly, the decreased peroxidase (POD) activity, consistent with down-regulation of expressed levels of POD1 and POD2, was observed in NAA-treated cuttings. Moreover, NAA resulted in a higher activity in polyphenol oxidase (PPO) compared with control cuttings. Collectively, the study highlighted that 0.54 mM NAA is efficient on rooting in hybrid aspen, and its effect on metabolic changes during rooting was discussed, which can provide valuable information for propagating hybrid aspen.