Alternanthera hirtula is a native plant from the Pampa biome in Brazil that is threatened with extinction due to loss of suitable habitats. Thus, the in vitro clonal propagation is a useful way to produce plants for the conservation of its germoplasm and to introduce it in nature. Shoots, obtained from wild plants were used as the source of nodal segments for in vitro culture.The effect of cytokinins (6-benzylaminopurine, kinetin or thidiazuron) and carbohydrates (sucrose, fructose or glucose) on plant production was tested. The best shoot multiplication was achieved on medium with 1.0 μM thidiazuron. Among carbohydrates tested, sucrose (30 or 40 g L-1) and glucose (20 g L-1) promoted better multiple shoot proliferation and plant development. Plants were successfully acclimatized and showed normal development in a greenhouse. Some plants were planted in botanical gardens and others were reintroduced into natural habitats. The proposed method could effectively be applied for clonal propagation and conservation of this important and endangered plant. Abbreviations: BAP: 6-benzylaminopurine, DW: Dry Weight, MS: Murashige and Skoog media, TDZ: Thidiazuron.