Although β-cryptogein gene expression was known to induce growth and phenolic metabolites accumulation in tobacco, however, the consequence on specialized metabolites other than phenolic has been scantly studied, and no report on the changes in primary metabolites is available. Tobacco (Nicotiana tabacum) hairy root cultures expressing β-cryptogein under CaMV 35S promoter were described here. GC–MS and HPLC–DAD based analyses of volatile and non-volatile metabolites including phenolic were carried out to study the variations in the levels of metabolites (both primary and specialized) in transgenic lines as compared to controls. Wall-bound phenolics and three forms of soluble phenolics (free, glycosyl-bound and ester-bound soluble phenolics) as well as chlorogenic acid contents were compared between cryptogein-cotransformed and control lines. In support of metabolite changes, semiquantitative expression analyses of selected genes of phenylpropanoid and terpenoid pathways viz. phenylalanine ammonia lyase (PAL), 4-coumaryl CoA ligase (4CL), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate synthase (DXS), were performed. Furthermore, activities of important enzymes (PAL and 4CL) involved in phenylpropanoid pathway were analyzed. Increase of phenolic compounds, fatty acid-derivatives, alkaloids (nicotine and nornicotine), benzenoids, sugars (sucrose, fructose, galactose and lactose) and a few metabolites originated from glycolysis pathway intermediates were noticed in Ri-crypt-transformed hairy roots (RCTHR) with decline in the contents of terpenoids and many amino acids (except proline). Increased gene expressions of PAL and 4CL along with enhanced activities of respective enzymes in RCTHR were supported the phenolics accumulation. Reduced expressions of HMGR and DXS genes were observed which supported the reduced amount of terpenoid metabolites. This study suggests a possible channelling of carbon pool towards phenylpropanoid/benzenoid pathway as well as lipoxygenase pathway and nicotine biosynthesis in tobacco hairy roots expressing a β-cryptogein gene. © 2020, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.