InCatharanthus roseuscell cultures, the terpenoid moiety is considered as the limiting factor in terpenoid indole alkaloid (TIA) production. The pathway of terpenoidal precursors in TIA is strongly linked with other terpenoid pathways, suggesting that TIA production might be limited by competition for the five-carbon (C5) precursors. This raises the question whether the stimulation of TIA production by certain signal molecules is due to a redistribution of C5 precursors between the associated terpenoid pathways and/or to a total increase of the precursor availability. To investigate the effect of a TIA-increasing signal molecule on C5 distribution, the cell suspension cultures ofC. roseuswere elicited with jasmonic acid (JA) and the metabolic changes of different terpenoid pathways were evaluated targeting on TIA (monoterpenoid; C10), carotenoids (tetraterpenoid; C40), and phytosterols (triterpenoid; C30). The chromatographic analyses showed that TIA and carotenoid levels almost doubled upon JA elicitation, while phytosterol levels remained constant if compared to the controls. Apparently, both TIA and carotenoid routes benefit from an increased C5 flow in the methyl-erythritol phosphate pathway, and potential export of C5 precursors to the cytosolic terpenoid routes, e.g., towards the phytosterols is minimal. However, the relative composition of individual compounds within each group remains similar when comparing elicited and control cells. This suggests that the increased production of TIA and carotenoid upon JA elicitation is predominantly caused by an increase of the precursor availability rather than due to a redistribution of C5 precursors between the associated terpenoid pathways. Furthermore, NMR-based metabolomics analysis showed a discrimination of JA-elicited and control cells between 24 and 72 h after treatments with significant changes in levels of strictosidine, malic acid, and sucrose. This study portrays metabolic alterations upon JA elicitation and channeling of C5 precursors in different terpenoid biosynthetic pathways, which provides a knowledge platform for developing strategies to engineer fluxes in a complex biosynthetic network in order to obtain high TIA-producingC. roseuscell lines.