For the implementation of commercial cultivation areas of heliconias, it is important to use pathogen-free seedlings and maintain the inherent characteristics of the hybrid plants to be cultivated. Thus, the objective of this work was to study a protocol for the in vitro propagation of the Heliconia bihai x Heliconia caribaea 'Jacquinii' hybrid and evaluate the plants survival rate during the acclimatization stage. Buds (explants) up to 5 mm long, originated from plantlets established in vitro were transferred to a ½ MS culture medium added by 0,5 mg L-1 of NAA combined with concentrations of 0.0; 0.5; 1.0; 1.5; 2.0 and 2.5 mg L-1 of 6-BA. Each treatment included 10 replications with one explant by replication, distributed at completely randomized design. After 100 days of growth under controlled conditions, rate of multiplication; plantlets length (cm) and cluster fresh and dry weight (g) were evaluated. Remaining plantlets were acclimatized in coconut fiber substrate to assess survival rate. In regards to the number of buds, the largest number of buds (four buds per explant) was verified in the absence of 6-BA, whereas in the presence of 6-BA, two to three buds per explant were observed. No significant differences were observed among 6-BA concentrations for plantlets length and cluster fresh weight. Plantlets survival rate during the acclimatization stage was 100%. Results from this study show that the MS culture medium added by 0.5 mg L-1 of NAA can be used for the in vitro propagation of the H. bihai x H. caribaea 'Jacquinii' hybrid