St. John's wort (Hypericum perforatum L.) is a medicinal plant used in the treatment of neurological disorders and has been recently shown to have anticancer potential. The principle medicinal components of St. John's wort are hypericin, pseudohypericin, and hyperforin. One of the problems associated with medicinal plant preparations including St. John's wort is the extreme variability in the phytochemical content, mostly due to environmental variability, and biotic and abiotic contamination during cropping. The current study was undertaken to transplant St. John's wort plants from in vitro bioreactors in a closed controlled environment system (CCES) with CO₂ enrichment for the optimized production of biomass and phytochemicals. The growth and levels of hypericin, pseudohypericin, and hyperforin in plants grown in CCES were compared with those of the greenhouse and in vitro-grown plants. The environmental parameters in the greenhouse were found to be variable whereas in the CCES these parameters were controlled. Generally, all the growth parameters and hypericin and pseudohypericin levels were significantly higher in the plants grown in the CCES. These results provide the first indication that growing St. John's wort plants under CO₂ enrichment in a closed environment system can enhance the biomass and medicinal contents. The adaptation of this growing system may be useful for the production of optimized products of St. John's wort and other medicinal species.