Abstract: Changes in soil microbial biomass C (MBC) and N (MBN) and their potential regulatory factors were investigated following long-term N fertilizer application since 2011 in a wheat-sweet potato rotation system. In the study, we aimed to provide a theoretical basis for the application of N fertilizers and to improve soil quality and ecological functions. Four N fertilization treatments were used: no fertilizer application (CK), no N-fertilizer application (PK), chemical N-fertilizer application (NPK), and chemical N-fertilizer combined with organic fertilizer application (NPKM). The soil MBC and MBN content, soil potential enzymes activities, and microbial C and N utilization efficiencies were investigated after the harvest of the two crops. Redundancy analysis and structural equation modeling were used to identify the potential biotic and abiotic factors that regulate MBC and MBN in the weakly acidic yellow-brown soils. The results showed that the contents of MBC and MBN and activities of sucrase and urease decreased in the N fertilization treatments. Compared with the NPK treatments, NPKM treatment increased the contents of MBC and MBN and the activities of sucrase and urease. Significantly higher contents of soil organic C (SOC), total N (TN), and mineral N (MN) were detected in the NPK and NPKM treated soils than in the CK and PK treated soils. However, long-term N fertilization significantly decreased the soil pH and efficiencies of microbial C and N utilization. Compared to the wheat season, the sweet potato season showed lower SOC and MN contents and higher MBN content and sucrase activity in the soils under all fertilization treatments. N fertilization strengthened the relationships between MBC and MBN and the sucrase and urease activities. Moreover, variations in the soil MBC and MBN contents and both sucrase and urease activities were regulated by soil pH, SOC and N content, and microbial C and N utilization efficiencies. Soil pH was the key factor driving the soil MBC content. Interactions were found between the activities of soil enzymes and the contents of MBC and MBN. In conclusion, long-term N fertilization decreased the MBC and MBN contents and the invertase and urease activities in yellow-brown soils. However, the combined application of chemical N fertilizers and organic fertilizer alleviated the decline in soil biological properties. The strong microbial C metabolism processes increased the MBN content in the wheat-sweet potato rotation system.