Adventitious root cultures were developed from Tripterygium regelii Sprague & Takeda and growth conditions optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f and four to the TPS-b subfamily. These genes were characterized by heterologous expression in a modular metabolic engineering system in E. coli. Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases and those belonging to the TPS-e/f family catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.