Vanda tricolor is an orchid endemic to Mount Merapi whose existence is threatened by the eruption of Mount Merapi which can occur at any time and the community has taken it directly from nature. Several attempts have been made to preserve the Vanda tricolor orchid, one of which is through in vitro culture. In vitro culture studies of Vanda tricolor have been carried out and large numbers of shoots have been obtained through multiplication and then raised by subculture. Plantlets obtained must be acclimatized to adapt and be returned to their original habitat. Growing medium and fertilizer application are important factors in the acclimatization process. This study aims to determine the best type of medium and fertilizer for acclimatization of Vanda tricolor orchids. This research was conducted using an experimental method in the preparation of a Factorial Completely Randomized Block Design. The first factor is the type of medium, namely coconut husk, moss and coconut husk + moss, the second factor is the type of fertilizer, namely without fertilizer, foliar fertilizer and liquid organic fertilizer. From the two treatment factors, 9 treatment combinations were obtained. Each treatment was repeated 3 times and each replication consisted of 3 samples. The results showed that there was a mutual influence between the type of medium and fertilizer. The coconut husk + moss is the best medium for acclimatization of Vanda tricolor orchids. Meanwhile, foliar fertilizer and liquid organic fertilizer compared to without fertilization, can increase the growth of Vanda tricolor orchids during acclimatization.