Resumo. In this study, an efficient method for regenerating plants from nodal cultures of seedlings was developed for Aspidosperma polyneuron. Mature seeds were surface-sterilized and embryos were germinated in Woody Plant Medium (WPM). Epicotyl and hypocotyl nodal segments, excised from 3-week-old in vitro -grown seedlings, were cultured in WPM medium supplemented with 6-benzyladenine (BA) (2.5, 5.0, and 10 μM), alone or combined with indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA) (0.5 μM) for culture initiation and three subcultures. For root induction, IBA (2.5, 5.0, and 10 mM) pulse treatments (15 minutes) were initially applied, followed by transfer to growth regulator-free WPM for five weeks. Regenerated plants were first transplanted to trays containing soil and Plantmax ® substrate (3:1) and later to polyethylene bags for acclimatization in a greenhouse. Hypocotyl explants exhibited superior shoot regeneration rate and rooting percentages compared with those of epicotyl explants. The highest numbers of shoots per explant (7 −8) were obtained at the third subculture when using the culture medium supplemented with 10 μM BA. When combined with BA, the addition of 0.5 μM IBA or NAA did not affect the regeneration of shoots. An IBA pulse treatment of 5 − 10 mM for 15 minutes induced 60% of rooting. The regenerated plants were acclimatized and successfully established in a greenhouse, with a 90% survival rate observed after three months. Based on the results of this study, the micropropagation of Aspidosperma polyneuron from in vitro seedlings is feasible and could be a useful tool for the conservation and propagation of this important endangered species.