Abstract Background Metabolic syndrome (MetS) is a multimorbid long-term condition without consensual medical definition and a diagnostic based on compatible symptomatology. Here we have investigated the molecular signature of MetS in urine. Methods We used NMR-based metabolomics to investigate a European cohort including urine samples from 11,754 individuals (18–75 years old, 41% females), designed to populate all the intermediate conditions in MetS, from subjects without any risk factor up to individuals with developed MetS (4–5%, depending on the definition). A set of quantified metabolites were integrated from the urine spectra to obtain metabolic models (one for each definition), to discriminate between individuals with MetS. Results MetS progression produces a continuous and monotonic variation of the urine metabolome, characterized by up- or down-regulation of the pertinent metabolites (17 in total, including glucose, lipids, aromatic amino acids, salicyluric acid, maltitol, trimethylamine N-oxide, and p-cresol sulfate) with some of the metabolites associated to MetS for the first time. This metabolic signature, based solely on information extracted from the urine spectrum, adds a molecular dimension to MetS definition and it was used to generate models that can identify subjects with MetS (AUROC values between 0.83 and 0.87). This signature is particularly suitable to add meaning to the conditions that are in the interface between healthy subjects and MetS patients. Aging and non-alcoholic fatty liver disease are also risk factors that may enhance MetS probability, but they do not directly interfere with the metabolic discrimination of the syndrome. Conclusions Urine metabolomics, studied by NMR spectroscopy, unravelled a set of metabolites that concomitantly evolve with MetS progression, that were used to derive and validate a molecular definition of MetS and to discriminate the conditions that are in the interface between healthy individuals and the metabolic syndrome.