Abstract Frailejones (Espeletia spp.) belong to the Asteraceae family and are endemic to South America. Many species have been severely affected, leading to population declines, including Espeletia occidentalis subsp. antioquensis, a Near Threatened species endemic to Colombia. We assessed the feasibility of in vitro mass propagation through organogenesis to support ex situ conservation and potential habitat reintroduction. Specifically, we evaluated the response of apical buds from germinated seedlings culture in Murashige and Skoog (MS) medium supplemented with cytokinins-auxins combinations for shoot induction and root formation. Initially, we observed that seed viability decreased during storage. In germination test, no significant differences were detected between gibberellic acid (GA3) concentrations; however, a significantly higher germination rate was achieved using moist cotton. The optimal conditions for in vitro multiplication were obtained in MS medium supplemented with 0.05 mg L−1 of 6-benzyl amino purine (BAP) and 0.5 mg L−1 indole-3-butyric acid (IBA), resulting in an average multiplication coefficient of 2.87 by the third subculture, with no significant differences among culture vessels. The highest rooting response was achieved with 3.0 mg L−1 IBA. A substrate mixture of topsoil and coconut fiber resulted in the highest survival rate (76.8%) and promoted subsequent development. This study presents the first report on the seed viability and in vitro propagation potential of E. occidentalis, including acclimatization strategies and prospects for habitat reintroduction. Between October 2022 and 2023, approximately 250 plants were successfully transplanted, achieving a 70% survival rate under páramo conditions.