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Ectomycorrhizal fungi form symbiotic relationships with a wide range of terrestrial plants, acquiring carbohydrates for themselves and promoting nutrient uptake in their host plants. However, some ectomycorrhizal fungi cannot effectively obtain the thiamine necessary for growth from their host or synthesize it themselves. Ectomycorrhizal fungi can recruit hypha-associated microorganisms, which play a vital role in promoting nutrient absorption and ectomycorrhizal root formation, ultimately colonizing within fruiting bodies to form a unique bacterial microbiota. In this study, non-targeted metabolomics and whole-genome sequencing were employed to investigate the colonization characteristics of the hyphae-associated bacterium Bacillus altitudinis B4 on the mycelial surface of ectomycorrhizal fungus Suillus clintonianus, as well as the synergistic promotion of thiamine synthesis and absorption by B. altitudinis B4 and the fungal mycelium, respectively. The results suggested that S. clintonianus first secreted ureidosuccinic acid and pregnenolone, recruiting the hyphae-associated bacterium B. altitudinis B4 to the mycelial surface. Subsequently, the ureidosuccinic acid secreted by S. clintonianus further stimulated B. altitudinis B4 to enhance thiamine production by increasing its biomass and upregulating the expression of related functional genes. Finally, S. clintonianus absorbed the thiamine secreted by the B. altitudinis B4, promoting fungal growth and increasing the colonization rate in association with Pinus massoniana. This study elucidates the thiamine acquisition mechanisms of ectomycorrhizal fungi, highlighting the critical role of bacterial partners in fungal nutrition and host-fungal interactions.