Abstract: Soil layers are exchanged during tillage practices, which may change the nitrogen (N) transformation process by affecting the physicochemical and biochemical properties of the soil. In this study, the effects of soil layers exchange on the nitrification and denitrification of lime concretion black soil, maize growth, and N utilization were studied to provide a theoretical basis for selecting reasonable tillage methods, reducing N loss, and improving N use efficiency in the southern region of the Huang-Huai-Hai Plain. In an artificial climate chamber, a normal soil layer distribution (0-35 cm of soil placed in a root box according to in situ soil layers) was used as the control treatment (CK). In-situ 0-10 cm and 10-20 cm soil layers were exchanged and placed in another group of root boxes, which were used as the soil layers exchange (SE) treatment group. A 20 μm nylon mesh was used to separate the rhizosphere and the bulk soil. To investigate the effects of soil layer exchange on soil N transformation, nitrification potential, respiration, denitrifying capacity, denitrification potential, physicochemical properties of the rhizosphere and bulk soil, as well as maize growth, and N use, total N content, and root morphology were investigated at the maize small trumpet stage. The results showed that maize N uptake in SE treatment was 8.9% lower than that of CK (P < 0.05). Soil layer exchange significantly affected the rhizosphere rather than the bulk soil, which reduced its nitrification potential by 13.5% (P < 0.05) and increased the denitrification capacity of the rhizosphere and the bulk soil by 36.6% (P < 0.05) and 8.4% (P < 0.05), respectively. Soil layers exchange increased the soluble organic carbon content of the rhizosphere and bulk soil by 11.7% (P < 0.05) and 5.2%, respectively. Correlation analysis showed that nitrification potential was significantly positively correlated with the abundance of ammonia-oxidizing bacteria (AOB, r=0.91^**), but was not significantly correlated with the abundance of ammonia-oxidizing archaea (AOA). Denitrification capacity was significantly positively correlated with soluble organic carbon and soil respiration (r=0.89^** and 0.93^**), but showed no correlation with nirK or nirS gene copy number. N uptake by maize plants was positively correlated with the nitrification potential of the rhizosphere and the total root surface area×AOB gene copy number (r=0.83^* and 0.86^*), but was significantly negatively correlated with denitrification capacity (r=-0.88^**). These results indicated that a decrease in the nitrification rate and an increase in the denitrification rate in lime concretion black soil could result in low N use efficiency by maize after soil layers exchange. The nitrification rate was driven more by AOB abundance. After soil layers exchange, soil soluble organic carbon was the key driving factor for denitrification capacity. Effective regulation of soil soluble organic carbon content is the key to improving crop nitrogen use efficiency under tillage conditions.