The arbuscular mycorrhizal fungi (AMF) effect on the plant metabolome is an actual question of plant biology. Its alteration during host plant development and at different phosphorus supplies is of special interest. The aim of this study was to evaluate the effect of Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler inoculation and/or phosphorus treatment on the root metabolome of Medicago lupulina L. subsp. vulgaris Koch at the first true leaf, second leaf, third leaf development stages, the lateral branching initiation, the flowering and the mature fruit stages. The assessment of metabolic profiles was performed using GC-MS. In total, 327 metabolites were annotated: among them 20 carboxylic acids, 26 amino acids, 14 fatty acids and 58 sugars. The efficient AM was characterized by the upregulation of the metabolism of proteins, carbohydrates and lipids, as well as an increase in the content of phosphates. The tricarboxylic acid abundance was generally lower during mycorrhization. Fourteen metabolic markers of the efficient AM symbiosis were identified. The lateral branching initiation stage was shown to have key importance. Long-lasting metabolomic profiling indicated variances in mycorrhization and Pi supply effects at different key stages of host plant development.