Ginger (Zingiber officinale), a member of the Zingiberaceae family, is a commonly available spice and medicinal plant. Ginger plantlets in tissue culture have not been commercialized because their ultimate success depends on the ability to successfully transfer the tissue culture plantlets from a controlled, aseptic environment to farm land while maintaining low costs and high survival rates. The survival rate of tissue culture plantlets transferred from a lab to soil is low, mainly because of differences in temperature, humidity conditions, biotic stresses, etc., which makes obtaining new seedlings challenging. This research aimed to optimize the protocol for acclimatization of tissue culture ginger plantlets. Rootless ginger tissue culture plantlets were initiated in seedling trays grown in a chamber maintained under a 16 h/day photoperiod with 60 μE m−2 s−1 of cool white light from fluorescent lamps at 25 ± 2 °C with an air relative humidity of 60 ± 5%, and the optimum substrate was vermiculite + peat (1:1 v/v) with MS + 0.5 mg/L NAA + 0.1% Metalaxyl-M·Hymexazol + 80% relative humidity (RH). After 40 days of culture, ex-vitro ginger seedlings had been successfully bred with a rooting rate of 100.0%, and the seedlings could be directly transplanted to the field.