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Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome and affect the uptake and transfer of mineral elements by forming a symbiotic relationship with plant roots. Nitrogen (N), as an important mineral element, can directly affect plant growth and development at different N levels. It has been confirmed that inoculation with AMF can improve the efficiency of N utilization by plants. However, there are still fewer reports on the dynamic relationship between arbuscular mycorrhizal and plant secondary metabolites at different nitrogen levels. In this experiment, the physiological indexes and genes related to saponin synthesis were determined by applying different concentration gradients of nitrogen to the medicinal plant P. polyphylla var. yunnanensis infested by AMF as the test material. It was found that nitrogen addition increased the biomass, chlorophyll content, and nutrient content of above- and below-ground plant parts and increased the content of saponin content of P. polyphylla var. yunnanensis to some extent, but AMF inoculation increased the saponin content of P. polyphylla var. yunnanensis more significantly. AMF inoculation also promoted the expression of genes related to the saponin synthesis pathway, including 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), squalene epoxidase 1 (SE1), and cycloartenol synthase (CAS), which is in according with the accumulation of saponin in plants. It also may increase the saponin content of AMF plants by altering the expression of P450s and UGTs related to saponin synthesis.