To clarify the transformation mechanism of dissolved organic nitrogen(DON) in vadose zone under alternating dry and wet conditions, an intensive agricultural region within the Dagu River Basin was used as the study area, and indoor experiments were performed to analyze dynamic changes in nitrogen content and its influencing factors (soil physical and chemical properties and microbial indicators) under varying frequencies of drywet cycles. The driving factors of nitrogen transformation were identified through redundancy analysis, and a path model was established based on the relationships among environmental factors, functional genes, and nitrogen transformation. The results show that DON undergoes rapid ammonification, with an accumulation of NO-3N during the dry period that subsequently transforms into NO-2N and NH+4N during the wet period, suggesting a dominance of nitrification processes during the dry period and denitrification processes during the wet period across all drywet alternation frequencies. With an increase in the number of drywet cycles in vadose zone from one to three times, the cumulative amount of NO-3N decreases during the dry period, while NO-3N depletion has a more pronounced decrease during the wet period, resulting in the residual NO-3N content (the mass of NO-3N in per kilogram of dry soil) increasing from 14.28 mg/kg to 34.16 mg/kg. Therefore, frequent drywet alternations increase the potential risk of DON exacerbating groundwater nitrate pollution. The values of relative abundance of the nitrifying genus Nitrospira in the dry period and the denitrifying genus Pseudarthrobacter in the wet period gradually decrease with the increase of the drywet alternation frequency, and the values of abundance of related functional genes also show a downward trend. Water content, redox potential, and dissolved organic carbon affect the expression of nitrogen transformationrelated genes, thereby indirectly regulating the transformation behavior of DON under drywet alternations.