This work aimed to evaluate the in vitro morphogenic response of adult explants of Bixa orellana and to establish a regeneration protocol from adult nodal and internodal explants from 16-year-old adult plants propagated by air-layering. For internodal segments the following treatments were used: T1, MS medium lacking growth regulators (MS0); T2, MS + 4.92 µM 6-I³,I³-dimetylalylamino-purine (2-iP); T3, MS + 4.56 µM Zeatin (ZEA); T4, MS + 4.44 µM 6-benzyladenine (BA); T5, MS + 4.54 µM Thidiazuron (TDZ), with ten repetitions for each treatment. Thirty days after establishment of INS, better regeneration rates were achieved with ZEA and TDZ, when compared to BA, 2-iP and MS0. For nodal segments the best results were obtained using the combination of 2iP and BA. In conclusion, the promotive effect of direct adventitious organogenesis from adult internodal segments or axillary shoot proliferation from adult nodal segments, in B. Orellana, is dependent on the type and concentration of cytokinin used. Histological analyses showed the presence of meristemoids in the regenerated tissue. This region was characterized by the typical organization of the vascular system into a cylindrical structure, with central xylem, and with fast proliferating and radially distributed cells. Our analyses also revealed that all budding activity had a vascular system connected to the original explant, confirming the organogenic pathway-related ontogeny. We suggest the use of MS medium supplemented with cytokinins ZEA or TDZ and 2iP or BA for regeneration of annatto from INS and NS, respectively, which will reduce the regeneration time of this species in vitro. The protocol could be further used in transformation systems applied to adult elite genotypes to improve bixin production. This is the first report on adventitious regeneration from adult explants of annatto.