Abstract <p>This research developed an effective protocol for micropropagation of <i>Ailanthus altissima</i> ‘Hongyechun’. Axillary shoots from dormant twigs dipped in water were sterilized with 70% ethanol for 15 s and with 0.1% HgCl₂ solution for 7 min, resulting in the survival rate of 71.3% and the contamination rate of 0%. The explants were then inoculated on Murashige and Skoog medium (MS medium) supplemented with 1 mg L⁻¹ 6-benzyladenine (6-BA), 0.1 mg L⁻¹ indole-3-acetic acid and 500 mg L⁻¹ polyvinylpyrrolidone (PVP), yielding the browning rate of 8.5% and the in vitro shoot induction of 79.8%. In vitro shoots were successfully multiplicated on modified MS medium (KNO₃ 1050 mg L⁻¹, NH₄NO₃ 925 mg L⁻¹, MgSO₄·7H₂O 285 mg L⁻¹, CaCl₂·2H₂O 300 mg L⁻¹) supplemented with 1.0 mg L⁻¹ 6-BA, 0.1 mg L⁻¹ indole-3-butyric acid (IBA) and 1000 mg L⁻¹ PVP, resulting in the shoot proliferation rate of 4.2 fold and the shoot vitrification rate of 4.5%. The most suitable medium for in vitro rooting was 1/2 MS medium supplemented with 1.5 mg L⁻¹ IBA, 1000&#xa0;mg L⁻¹ PVP, 2% sucrose and 0.6% agar, resulting in the root induction rate of 85.8%. The suitable acclimatization medium was a mixture of vermiculite, peat, carbonized rice husk, fermented sawdust and coal ash (at a volume ratio of 2 : 2 : 4 : 1 : 1). About 85% of the in vitro plantlets were acclimatized in glasshouse after 35 days. The rooted and acclimatized plants were successfully transferred to soil after light-hardening under sunlight for 5–7 days resulting in 93% survival.</p>