1. Soil protist plays a key role in ecological functions through predation and parasitism. However, little is known about how nitrogen (N) deposition and seasonal variations influence soil protist function in forest soils.

2. Here, we assessed firstly the impacts of N deposition (control, 50 kg N ha^-1 yr^-1, 100 kg N ha^-1 yr^-1, and 150 kg N ha^-1 yr^-1) on the functional composition of the soil protist community in summer and winter, using amplicon sequencing of environmental DNA from a subtropical natural forest.

3. We found that soil protists were dominated by consumers (42.6–51.6%), followed by parasites (32.9–40.9%) and phototrophs (3.2–13.1%), implying a predominant role of consumers and potential top-down effects on the other trophic groups in subtropical forest soils. The functional composition of soil protists was greatly influenced by N deposition, but these responses were dependent on seasonal variations. The diversity of phototrophs was lower in summer than in winter. Instead, an opposite pattern was observed for consumers, resulting in a significantly higher protist diversity in summer than in winter, which indicates a greater sensitivity of soil protists to seasonal variations. Furthermore, low and high N deposition simplified the structural complexity of soil protist communities, suggesting a nonlinear response of the protist structural stability to N deposition.

4. Synthesis and applications. This study provides unprecedented evidence that season variation plays an important role in regulating responses of soil protist functional composition to N deposition, and highlights the nonlinear effects of rising N deposition levels on the soil food web.