The phenylpropanoid biosynthetic pathway represents one of the most important secondary metabolite biosynthetic pathways in plants. Although being investigated for many years, the role and regulation of many phenylpropanoids in planta remains unknown. This is notably the case with lignans, whose beneficial health effects are recognized in terms of antioxidant, bacteriostatic, phytoestrogenic and cancer chemoprotective effects. In order to get a better insight into the biosynthesis of phenylpropanoids, RNAi mediated pinoresinol-lariciresinol reductase 1 (PLR 1) mutants were characterized using a metabolomic approach. PLR 1 is the enzyme responsible for the biosynthesis of the main lignan in flax (+)-secoisolariciresinol diglucoside ((+)-SDG). The used analytical techniques were NMR and LC-MS. The mutant lines have shown a significantly different composition regarding 19 metabolites. It has been observed that the loss of PLR 1 function significantly affected the content of secondary metabolites (lignans, phenolic acids and cyanogenic glycosides) but primary metabolites as well (amino acids and raffinose). A drastic decrease in the (+)-SDG content was observed as well as an increase in the contents of glucosilated forms of lariciresinol and pinoresinol. The PLR 1 mutants showed the loss of ability to accumulate the lignan (+)-SDG instead of which they incorporate the glucosilated forms of lariciresinol and pinoresinol in the lignan macromolecule. The observed effects may correspond to a redirection of lignan flux via PLR 2, an enzyme already characterized in flax seed, and/or support the hypothesis of the presence of PLR 3, on which some indications have recently been reported.