Micropropagation is one technology to propagate endemic species of the Ferruginous Rupestrian Grasslands when in vitro genetic conservation is sought. The present study aimed to assess the breaking of dormancy, in vitro establishment, multiplication, elongation, rooting, and acclimatization of Mimosa calodendron from culture in a temporary immersion bioreactor system. The seeds used for the experiments were from plants originating from the Ferruginous Rupestrian Grasslands. The percentage of contamination, oxidation, unresponsive seeds, germination, number of buds per explant, shoot length, senescence, percentage of adventitious rooting, and acclimatization were assessed. The breaking of dormancy was most successful by mechanical scarification (80% germination). Immersion in sodium hypochlorite for 5 minutes was the most efficient treatment for in vitro establishment (90%). For the in vitro multiplication and elongation phase, the use of liquid culture medium from cultivation in a temporary immersion bioreactor was the most suitable for the characteristics number of buds per explant (2.55 buds), vigor (1.1), oxidation (1.3) and senescence (1.3) according to the score’s scale. Regardless of the in vitro cultivation method, the percentages of rooting and acclimatization were satisfactory, and it was possible to obtain complete plants in 190 days.