Taxus stem barks can be used for paclitaxel extraction. However, the composition of taxoids across the whole stem and stem tissue-specificity of paclitaxel biosynthesis-related enzymes remain largely unknown. Cultivated Taxus media trees were used for chemical composition and protein analyses of major stem tissues using an integrated metabolomic and proteomic approach, and the role of TmMYB3 in paclitaxel biosynthesis was investigated. The metabolomic landscape analysis showed differences in stem tissue-specific accumulation of metabolites. Phytochemical analysis revealed that paclitaxel highly accumulated in the phloem. Ten key enzymes involved in paclitaxel biosynthesis were identified, most of which were predominantly produced in the phloem. The full-length sequence of TmMYB3 and partial promoter sequences of five paclitaxel biosynthesis-related genes were isolated. Several MYB recognition elements were found in the promoters of TBT, DBTNBT and TS. Further in vitro and in vivo investigations indicated that TmMYB3 was involved in paclitaxel biosynthesis by activating the expression of TBT and TS. Differences in the taxoid composition of different stem tissues suggested that the whole stem of T. media has potential for biotechnological applications. Phloem-specific TmMYB3 plays a role in the transcriptional regulation of paclitaxel biosynthesis, and may explain the phloem-specific accumulation of paclitaxel.