Astrophytum myriostigma is an important ornamental cactus with a significant international trade demand, often collected from the wild. The multiplication of A. myriostigma by traditional propagation methods is insufficient to meet the current market demand. This study aimed to establish a protocol for micropropagation of A. myriostigma through areoles activation. Apical and basal explants from plants cultivated in polyhouse were incubated on Murashige and Skoog (MS) medium with 1-12 µM of N⁶-benzyladenine (BA), 6-Furfurilaminopurine (Kinetin; Kin), or 1-Phenyl-3-(1,2,3,-thiadiazol-5-yl) urea (Thidiazuron; TDZ) for shoot regeneration. Of the two A. myriostigma explants studied, apical explants were found to be the best for shoot production. Among the three cytokinins investigated, Kin was most powerful in inducing shoots from both explant types by areoles activation. Supplementation of 0.5 or 1.0 µM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or α-naphthaleneacetic acid (NAA) to the optimal shoot induction MS medium containing 2 µM Kin improved the regeneration capacity of apical and basal explants of A. myriostigma. The best shoot production was attained on MS nutrient medium enriched with 2 µM Kin and 1 µM IBA. The highest rooting percentage (100%), maximum root numbers (14.7), and longest root length (6.1 cm) were obtained on MS medium supplemented with 4 µM of IBA. Plantlets were well acclimatized in the greenhouse with a survival rate of 93%.