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In agricultural production, intercropping is a widely used system with many benefits. Lupin ( Lupinus angustifolius  L.) is a legume that contains a large variety of plant secondary metabolites, which have multiple functions in the plant, e.g. signalling, nodulation and stress response. An untargeted metabolomics approach was applied to investigate how the metabolome of lupin was affected by intercropped barley ( Hordeum vulgare  L.). The only primary metabolite of lupin affected by intercropping was tryptophan. Several secondary metabolites were affected by intercropping in lupin, and five flavonoids were annotated hereof. The flavonoid levels were increased, and tryptophan levels decreased in lupin when intercropped. Two flavonoids are prenylated, and prenylated flavonoids are believed to play a role in the plant’s stress response. Furthermore, flavonoids are involved in plant defence and the nodulation process. Thus the present flavonoids may affect regulation of lupin N 2 -fixation activity. HighlightsIntercropping caused a change in the secondary metabolites of  Lupinus angustifolius  L.Prenylated flavonoids were generated in  Lupinus angustifolius  L. as a response to intercroppingTryptophan was the only identified primary metabolite affected by intercropping Intercropping caused a change in the secondary metabolites of  Lupinus angustifolius  L. Prenylated flavonoids were generated in  Lupinus angustifolius  L. as a response to intercropping Tryptophan was the only identified primary metabolite affected by intercropping

Intercropping of Hordeum vulgare L. and Lupinus angustifolius L. causes the generation of prenylated flavonoids in Lupinus angustifolius L.