Abstract: The Lanmaoa asiatica, a precious wild edible ectomycorrhizal fungus found in Yunnan, China, exhibits extremely slow mycelial growth under pure cultivation conditions. In order to further understand the substances associated with the mycelial growth of Lanmaoa asiatica under pure cultivation, this study employed three metabolomic techniques: proton nuclear magnetic resonance (1H-NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). These techniques were used to analyze the small molecular differences between the mycelium (J35) and the attached tissue blocks (Z35) after 35 days of pure cultivation. The research results showed that there were 232 differential substances between Z35 and J35. Among them, 131 substances including glutamic acid were significantly upregulated, while 12 substances including 4-aminobutyric acid, succinic acid, and hydrastine were only detected in the mycelium. The 131 upregulated substances were enriched in 26 pathways, with the most significant impact observed in pathways related to alanine, aspartate, and glutamate metabolism, butyrate metabolism, and pathways involving glycine, serine, and threonine metabolism. Glutamic acid acted as a hub connecting these three pathways, while 4-aminobutyric acid, succinic acid, and hydrastine were identified as key substances in the butyrate metabolism pathway. The butyrate metabolism pathway served as the core pathway, through which glutamic acid, 4-aminobutyric acid, succinic acid, and hydrastine entered the central hub of nutrient metabolism (TCA cycle), providing nutritional substances for the growth of the mycelium. The 232 differential substances, including glutamic acid, may play a regulatory and promoting role in the growth of the mycelium. This study provides a theoretical basis for the artificial cultivation of Lanmaoa asiatica.