Living plant collections play a crucial role in ex situ conservation, but their conservation value hinges on assessment of their composition, data quality and on strategic planning for optimization of the collection that takes into account species diversity, conservation status and allocation of resources. Cactaceae is one of the most threatened plant families globally and has a broad distribution in Brazil, with two of the seven centres of diversity for this group and a total of 276 known species. Climate change could escalate the threat to cacti, potentially placing 90% of species at risk by 2050. We conducted a case study of Cactaceae in the living Cactarium collection of the Rio de Janeiro Botanic Garden, evaluating the diversity of the collection and recommending strategic actions to enhance its conservation value. In 2022, we inventoried all specimens in the collection and cross-referenced them with the institutional specimen registry system, Jabot. The collection contains 156 cacti species that naturally occur in Brazil (57% of the total), including 43 species categorized as threatened on the Brazilian Red List and 57 categorized as threatened on the IUCN Red List. We recommend research to map cacti species that are threatened but not covered by in situ conservation in the country, list the priority species for acquisition by the collection, and make recommendations that will facilitate improved contribution of the Rio de Janeiro Botanic Garden to the conservation of the Brazilian flora.