Abstract: The clay layer in the vadose zone with layered structure in soils has an important effect on blocking the pollutants which will enter into the groundwater. At present, there is insufficient knowledge about the effects of the thickness of the clay layer on the migration process of nitrate nitrogen (NO3--N). Nitrate nitrogen is more prone to leaching through the 'up thick and down thin' ( the up layer of soil with larger particles and the down layer of soil with smaller particles) vadose zone with layered structure, which can cause a higher risk of nitrate nitrogen pollution to groundwater. In this study, we set up four groups soil columns with 40 cm height, the types of four groups soil columns included "up sand and down clay" and full filling with clay. The ratio of thickness (between sand and clay layer) was 3:1, 1:1, and 1:3, and the last type soil column had 40 cm clay. The content of the study in the leaching experiment under the condition of stable water head and stable concentration of nitrate nitrogen with infiltration, which included infiltration process of nitrate nitrogen solution, the characteristics of accumulation and denitrification of nitrate nitrogen in soil columns. In order to clarify the effects of the clay layer thickness on the migration process of nitrate nitrogen in the layered vadose zone, the soil columns with different thickness of clay layer. The results showed that the infiltration process of solution could be divided into two stages, namely, the relationship between the cumulative infiltration capacity with the wetting front transport depth and the infiltration time gradually changed from nonlinear to linear when the wetting front crossing the sand-clay layer interface. Meanwhile, the cumulative infiltration capacity decreased significantly with the increasing in the thickness of clay layer in the soil columns (P<0.05). When the thickness of the clay layer in the soil column reached 40 cm, the effect of blocking on the nitrate leaching of nitrogen was significantly stronger than other soil column which was with the clay layer of 10-30 cm. During the leaching experiment, a water retention layer was formed at the interface of the sand-clay layer, which made the cumulative values of nitrate nitrogen and nitrite nitrogen in the clay layer at the interface were both large, while the accumulation of nitrate nitrogen and nitrous nitrogen decreased with the increasing of depth in clay layer in the same soil column. When the difference of thickness in clay layer was not less than 20 cm during different soil columns, the accumulation of nitrate nitrogen in the soil column was significantly different (P<0.05). The soil column which thickness of clay layer was 40 cm had the largest amount of nitrate nitrogen used in denitrification [(0.15±0.05) g] was significantly higher than the soil column with 10-30 cm in the thickness of clay layer (P<0.05), which indicated that when the thickness of clay layer reached a certain degree (such as 40 cm), it not only blocked nitrate nitrogen, but also affected the amount of denitrification. Occurrence had a significant impact which could play an important role in preventing the leaching of nitrate nitrogen into groundwater. This study can provide a reference for the management of farmland fertilization and groundwater protection under soil with layered vadose zone conditions.