Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many researchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21 d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplanting ex vitro compared with those cultured under Pm conditions.