Abstract: Clarifying the effects of Chinese milk vetch (CMV) incorporation and substitution of conventional nitrogen fertilizers with slow-release nitrogen fertilizers on soil organic carbon (SOC) fractions, carbon pool management index (CPMI) and the maintenance/increase of rice grain yield, can provide a theoretical basis for formulating application schemes of slow-release nitrogen fertilizers that are conducive to SOC sequestration and the sustainable utilization of paddy fields. In this study, the effects of CMV incorporation and substitution of conventional nitrogen fertilizers with slow-release nitrogen fertilizers on SOC, labile organic carbon (LOC) and CPMI were studied quantitatively in rotation systems of rice-wheat and rice-CMV in the middle and lower reaches of the Yangtze River Basin, and even investigated their effects on the maintenance/increase of rice grain yield. The experiment adopted a two-factor completely randomized block design, with the main factors being the crop rotation systems: rice-wheat (W-R) and rice-CMV (CMV-R). The secondary factor was the nitrogen fertilizer application methods: conventional nitrogen fertilizer (CN), slow-release nitrogen fertilizer substitution for conventional nitrogen fertilizer based on the same nitrogen input (SRN), 10% nitrogen reduction based on SRN during wheat growth period and 20% nitrogen reduction based on SRN during rice growth period (T1), 20% nitrogen reduction based on SRN during wheat growth period and 40% nitrogen reduction based on SRN during rice growth period (T2), 30% nitrogen reduction based on SRN during wheat growth period and 60% nitrogen reduction based on SRN during rice growth period (T3). Compared with the W-R, the rice yield of the CMV-R significantly increased by 2.0%, while the annual net income decreased by 20.4%. The contents of total labile organic carbon (TLOC), high labile organic carbon (HLOC), and medium labile organic carbon (MLOC) as well as CPMI increased significantly by 5.2%, 6.0%, 5.1%, and 6.6% respectively. However, there was no significant difference in contents of SOC, low labile organic carbon (LLOC), and no labile organic carbon (NLOC) between W-R and CMV-R. Compared with the CN, the rice yields of SRN and T1 increased significantly by 12.6% and 7.3%, and the annual net income increased by 38.8% and 20.7% respectively, while the rice yield of T3 decreased by 9.4%. The contents of TLOC, HLOC and CPMI in SRN increased significantly by 7.1%, 6.9% and 9.4%, respectively, relative ti the CN, while there was no significant difference for SOC content between CN and SRN. Meanwhile, there were no significant differences in contents of SOC, TLOC, HLOC, MLOC, LLOC and NLOC as well as CPMI between T1 and CN, while they decreased significantly under T2 and T3 relative to the CN. Based on the crop rotation system and nitrogen fertilizer application methods, the rice yield, SOC, TLOC, HLOC, MLOC contents and CPMI of SRN under CMV-R were all higher than those of the other treatments, followed by the SRN under W-R and the T1 under CMV-R. In addition, no significant difference was found for the rice yield, SOC, TLOC, HLOC, MLOC contents and CPMI between T1 under CMV-R and SRN under W-R. Therefore, in the rice-growing areas in the middle and lower reaches of the Yangtze River Basin, adopting rice-Chinese milk vetch rotation system combined with substitution of conventional nitrogen fertilizers with slow-release nitrogen fertilizers (SRN), and even under 20% nitrogen reduction based on SRN is conducive to soil organic carbon sequestration and the sustainable utilization of paddy fields, which is of great significance for the sustainable production of agriculture in this region