The aim of this investigation was to acclimatize the ginger (Zingiber officinale Rosc.) plantlets cultured photoautotrophically in vitro under different RH and CO2 concentrations and subsequent ex vitro adaptation. Plantlets acclimatized in vitro under medium or high RH (80±5 or 95±5% RH) with 1,000±100 µmol mol-1 (CO2-enrichment) conditions possessed significantly higher Relative Water Content (RWC) than those acclimatized in vitro under low RH (65±5% RH) with 450±100 µmol mol-1 (CO2 non-enrichment) conditions. The RWC of acclimated plantlets was positively related to maximum quantum yield of PSII photochemistry (Fv/Fm) (r = 0.77), quantum efficiency of PSII (FPSII) (r = 0.89) and stomatal conductance (G) (r = 0.99), while negatively related to transpiration rate (r = 0.98). Moreover, the total chlorophyll concentration was closely related to FPSII (r = 0.77), lead to increase net photosynthetic rate (NPR) (r = 0.87). As well as, the intracellular CO2 concentration (Ci) and WUE of acclimatized plantlet positively related to NPR (r = 0.96 and r = 0.85, respectively), resulting in growth promotion (r = 0.99), as defined by the parameters of leaf area, fresh weight, dry weight, root number and root length. Five days after transplantation, the WUE, G, Fv/Fm and FPSII of ginger plantlets acclimatized in vitro under high RH with CO2-enriched conditions were significantly higher than those acclimatized in vitro under low RH and without CO2 enrichment, while E and transpiration ratio (Tr) were significantly lower. The plantlets acclimatized under high RH with CO2-enrichment conditions showed the highest adaptive abilities and WUE, resulting in the highest survival percentage (90-100%) after transplantation to ex vitro.