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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 N2-fixation activity., 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 N2-fixation activity.