The objective of this study was to develop an in-vitro acclimatization technique through relative humidity reduction for direct transplanting of woody species to ex-vitro. The survival percentage, growth efficiency and physiological characteristics of in-vitro acclimatized plantlets and ex-vitro adaptation were then evaluated. Thai neem (Azadirachta siamensis Val.) was selected for this research as it is a species with high potential for regenerating soils with high salt content. Twenty-eight day-old plantlets were transferred to environmental control chambers under controlled relative humidity (RH), which are 655% and 955% for low and high RH respectively, in order to acclimatize plantlets for 42 days. It was found that leaf water content, chlorophyll content and net photosynthetic rate (NPR) of plantlets acclimatized under low RH conditions were significantly higher than those acclimatized under high RH conditions (control). The relationship of leaf water content and chlorophyll content with NPR directly affects growth efficiency, leaf area and dry weight, of plantlets. After transplanting, the water relation system [water use efficiency (WUE), stomatal conductance (G) and transpiration rate (E)] and photosynthetic system [NPR, maximum quantum yield (F v /F m ) and quantum efficiency of photosystem II ( PSII )] were measured at days 0, 1, 3 and 5. The plantlets acclimatized under low RH conditions showed higher physiological adaptation in G, NPR, F v /F m and PSII , except E, than those acclimatized under high RH conditions. The plantlets acclimatized under low RH conditions protected water loss through the leaf tissues by the function of stomata closure as indicated by low E values. Consequently, the survival percentage of plantlets acclimatized under low RH conditions was higher by a factor of 2.3 than those acclimatized under high RH conditions. In-vitro plantlets acclimatized by reducing RH produced vigorous plantlets and directly transplanted to ex-vitro with a high survival rate of 87.5%.