© 2020 John Wiley & Sons, Ltd. Introduction: Nicotiana tabacum is a plant model intensively used in the bio-engineering pharmaceutical industry as a platform to produce drugs and therapeutic agents. Currently, no information regarding the non-targeted metabolome of transgenic tobacco containing recombinant regulatory sequences is available. Objective: To compare the proton nuclear magnetic resonance (1H-NMR) metabolomics profiling of a recombinant Nicotiana tabacum strain containing a promoter of a sesquiterpene cyclase from Capsicum annuum driving GUS expression, versus wild-type samples. Methodology. The non-targeted 1H-NMR metabolome was obtained and processed by principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The differential metabolites were quantified by quantitative NMR. Results: PCA and OPLS-DA revealed 37 metabolites including 16 discriminant compounds for transgenic samples. Ethanol (0.4 mg g-1), the main differential compound, was exclusively detected in transgenic tobacco; however, high levels of formate (0.28 mg g-1) and acetate (0.3 mg g-1) were simultaneously observed in the same group of samples. Cembratriene-4,6-diol, an antitumour and neuroprotective compound, and capsidiol, a known phytoalexin, increased by about 30% in transgenic samples. In addition, the endogenous levels of the antioxidant caffeoylquinic acid isomers increased by 50% in comparison to those of wild-type tobaccos. Conclusion: Our results support the occurrence of metabolic differences between wild type and transgenic tobacco containing a promoter of a Capsicum sesquiterpene cyclase gene. Interestingly, the recombinant transgenic strain studied accumulated high amounts of added value compounds with biological activity.