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Conventional in vitro propagation may affect normal morphophysiological characteristics and survival during the acclimatization of commercial plants, such as Etlingera Elatior (Jack) R.M. Smith. CO 2 enrichment, gas-permeable membranes, and reduced carbohydrate concentration in culture media can be used to mitigate these effects. Here, we aimed to determine the impact of photomixotrophic and photoautotrophic growth on morphophysiological responses of E. elatior and its survival during ex vitro acclimatization. In vitro-established plants were placed in flasks containing semisolid MS medium with 0.54 µM α-naphthalene acetic acid and sucrose (0 or 15 g L −1 ). The flasks were sealed with lids with or without orifices covered by gas-permeable membranes and kept for 45 days under forced-air ventilation (360 or 1000 µmol −1 CO 2 ). Growth variables, anatomical and physiological characteristics, and survival rate were evaluated. Overall, 1000 µmol mol −1 CO 2 , gas-permeable membranes, and 15 g L −1 sucrose resulted in plants with higher fresh and dry mass, higher level of photosynthetic pigments, as well as more developed secondary ribs in the leaves, prominent midribs, highly organized and differentiated chlorenchyma cells (palisade and spongy parenchyma), more developed vascular bundles, and intercellular gaps were reduced, compared to conventionally in vitro-propagated plants. Moreover, these conditions promote morphophysiological responses and favored survival of E. elatior plants (75%) when transferred to ex vitro conditions.