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Zenia insignis Chun is a large, fast-growing deciduous tree. In this study, we successfully developed a reliable and efficient protocol for the regeneration of fertile plants via callus induction from leaf segments of young Z. insignis seedlings. The best results were obtained with a medium containing 11.00 μM 6-benzyladenine (6-BA), 1.20 μM indole-3-butytric acid (IBA), and 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), which yielded morphogenic callus within 2 weeks at a frequency of 62.23%. We tested the effect of IBA alone and in combination with 6-BA on the bud differentiation response of Z. insignis callus. Shoots differentiated normally when cultured on differentiation medium containing 6.00 μM 6-BA and 1.20 μM IBA. Regenerated buds elongated successfully in medium containing 1.20 μM gibberellic acid (GA3). The elongated shoots were then transferred to Murashige and Skoog basal medium supplemented with various combinations of naphthalene acetic acid (NAA) for root induction; well-developed roots were achieved on MS basal medium supplemented with 0.01 μM NAA at a rooting rate of 89.23%. Rooted plantlets were successfully acclimatised to a greenhouse at a survival rate exceeding 90.00%.