Arbuscular mycorrhizae (AM) fungi form symbiotic associations with plant roots, providing nutritional benefits and promoting plant growth and defenses against various stresses. Metabolic changes in the roots during AM fungal colonization are key to understanding the development and maintenance of these symbioses. Here, we investigated metabolic changes in the roots of peanut (Arachis hypogaea L.) plants during the colonization and development of AM symbiosis, and compared them to uncolonized roots. The primary changes during the initial stage of AM colonization were in the contents and compositions of phenylpropanoid and flavonoid compounds. These compounds function in signaling pathways that regulate recognition, interactions, and pre-colonization between roots and AM fungi. Flavonoid compounds decreased by 25% when the symbiosis was fully established compared to the initial colonization stage. After AM symbiosis was established, general metabolism strongly shifted toward the formation of lipids, amino acids, carboxylic acids, and carbohydrates. Lipid compounds increased by 8.5% from the pre-symbiotic stage to well-established symbiosis. Lyso-phosphatidylcholines, which are signaling compounds, were only present in AM roots, and decreased in content after the symbiosis was established. In the initial stage of AM establishment, the content of salicylic acid increased two-fold, whereas jasmonic acid and abscisic acid decreased compared to uncolonized roots. The jasmonic acid content decreased in roots after the symbiosis was well established. AM symbiosis was associated with high levels of calcium, magnesium, and D-(+)-mannose, which stimulated seedling growth. Overall, specific metabolites that favor the establishment of AM symbiosis were common in the roots, primarily during early colonization, whereas general metabolism was strongly altered when AM symbiosis was well-established. In conclusion, specialized metabolites function as signaling compounds to establish AM symbiosis. These compounds are no longer produced after the symbiosis between the roots and AM becomes fully established.