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Dissolved oxygen (DO) stratification is a natural phenomenon in lakes, which potentially influences nitrogen (N) biogeochemical cycle. However, the specific effects of DO stratification on N speciation and transformation behaviors in different water layers are still poorly understood. Here, we reported that DO stratification remarkably influenced N species and transformation pathways in different water columns by high frequency sampling during summers in Longjing Lake, China. Results showed that DO stratification in the lake created three water layers: epilimnion (1-3m), oxycline (4-11m), and hypolimnion (12-20m). In the epilimnion, N speciation was mainly controlled by phytoplankton assimilation and organic N dominated in this layer. Oxycline was the major place for N transformations and had the most notable N removal capacity (714kgN from June to August). In the hypolimnion, NH4+ was the major N species, and sediment release contributed nearly 85% hypolimnetic NH4+. Furthermore, approximately 8kg of dissolved N2O was also accumulated in the hypolimnion, contributing similar to 70% of N2O in the whole lake. Overall, our results indicated that DO stratification caused the shifts in N speciation and transformation behaviors among different water columns, which may have a great implication for lake managements for providing separated protection strategies from different water depths.