Long-term manure application is recognized as an efficient management practice to enhance soil organic carbon (SOC) accumulation and nitrogen (N) mineralization capacity. A field study was established in 1979 to understand the impact of long-term manure and/or chemical fertilizer application on soil fertility in a continuous maize cropping system. Soil samples were collected from field plots in 2012 from 9 fertilization treatments (M0CK, M0N, M0NPK, M30CK, M30N, M30NPK, M60CK, M60N, and M60NPK) where M0, M30, and M60 refer to manure applied at rates of 0, 30, and 60 t ha(-1) yr(-1), respectively; CK indicates no fertilizer; N and NPK refer to chemical fertilizer in the forms of either N or N plus phosphorus (P) and potassium (K). Soils were separated into three particle-size fractions (2000-250, 250-53, and <53 μm) by dry- and wet-sieving. A laboratory incubation study of these separated particle-size fractions was used to evaluate the effect of long-term manure, in combination with/without chemical fertilization application, on the accumulation and mineralization of SOC and total N in each fraction. Results showed that long-term manure application significantly increased SOC and total N content and enhanced C and N mineralization in the three particle-size fractions. The content of SOC and total N followed the order 2000-250 μm > 250-53 μm > 53 μm fraction, whereas the amount of C and N mineralization followed the reverse order. In the <53 μm fraction, the M60NPK treatment significantly increased the amount of C and N mineralized (7.0 and 10.1 times, respectively) compared to the M0CK treatment. Long-term manure application, especially when combined with chemical fertilizers, resulted in increased soil microbial biomass C and N, and a decreased microbial metabolic quotient. Consequently, long-term manure fertilization was beneficial to both soil C and N turnover and microbial activity, and had significant effect on the microbial metabolic quotient.