<p>Withanolides are bioactive triterpenoids that require isoprene units viz., isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as precursor molecule for their biosynthesis, mediated via two independent routes, the plastidial methyl erythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathway. Specific inhibitor of rate limiting enzymes i.e., 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) of the MEP pathway i.e. fosmidomycin and the enzyme HMG CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) of MVA pathway i.e. mevinolin were used to perturb the metabolic flux and the effect on both primary and secondary metabolism were analysed. Foliar application of fosmidomycin and mevinolin resulted in a significant reduction of 33–40% in DXR enzyme activity and ~ 33% in HMGR enzyme activity. Also, the fosmidomycin mediated inhibition of the DXR activity significantly induced the HMGR activity and vice-versa with mevinolin. Further, an inhibition of any one pathway MEP or MVA caused a stimulation of the other for maintaining the metabolic homeostasis, while inhibition of both the MEP and MVA pathways adversely affected the photosynthetic characteristics and the growth parameters. Under the MEP pathway inhibition, up to 50% reduction in the withaferin A content was evidenced. Further, the radiotracer (¹⁴C) studies executed to assess the quantum of total labelled carbon-metabolite accumulation, including withanolides, in the roots under the exposure of MEP and MVA pathway inhibitors, confirm the above findings, though not in an absolute manner. The present study indicates that both MEP and MVA pathways regulate the withanolide production in <i>W. somnifera.</i> wherein the MVA pathway appears to be the major supplier of IPP for the withanolide biosynthesis, while the MEP pathway exerts an indirect control on IPP availability through photosynthesis and carbon assimilation.</p> Graphical abstract <p></p>