Golden berry (Physalis peruviana L.) is an exotic fruit exported from Colombia to different countries around the world. A review of the literature tends to demonstrate a hypoglycaemic effect with an improvement in insulin sensitivity after oral ingestion of fruit extracts in animal models. However, little is known about their potential effects in humans, and very little is known about the mechanisms involved. This study aimed at identifying discriminant metabolites after acute and chronic intake of golden berry. Method: An untargeted metabolomics strategy using high-performance chemical isotope-labelling LC-MS was applied. The blood samples of eighteen healthy adults were analysed at baseline, at 6 h after the intake of 250 g of golden berry (acute intervention), and after 19 days of daily consumption of 150 g (medium-term intervention). Results: Forty-nine and 36 discriminant metabolites were identified with high confidence, respectively, after the acute and medium-term interventions. Taking into account up- and downregulated metabolites, three biological networks mainly involving insulin, epidermal growth factor receptor (EGFR), and the phosphatidylinositol 3-kinase pathway (PI3K/Akt/mTOR) were identified. Conclusions: The biological intracellular networks identified are highly interconnected with the insulin signalling pathway, showing that berry intake may be associated with insulin signalling, which could reduce some risk factors related to metabolic syndrome. Primary registry of WHO.