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 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 crop yield, economic benefit, 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, and even investigated their effects on the maintenance/increase of rice 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 application (CN), slow-release nitrogen fertilizer substitution for conventional nitrogen fertilizer based on the same nitrogen input (SRN), 20% nitrogen reduction in rice season and 10% nitrogen reduction in wheat season based on SRN (T1), 40% nitrogen reduction in rice season and 20% nitrogen reduction in wheat season based on SRN (T2), 60% nitrogen reduction in rice season and 30% nitrogen reduction in wheat season based on SRN (T3). Compared with the W-R rotation system, the rice yield of CMV-R rotation system significantly increased by 2.6% (P<0.05), while the annual net income significantly decreased by 20.4% (P<0.05); the contents of total labile organic carbon (TLOC), highly labile organic carbon (HLOC), and medium labile organic carbon (MLOC) as well as CPMI of CMV-R rotation system increased significantly by 5.2%, 6.0%, 5.1%, and 6.6% (P<0.05), respectively. Compared with the CN treatment, the rice yields under SRN and T1 treatments increased significantly by 12.6% and 7.3% (P<0.05), and the annual net income increased significantly by 38.8% and 20.7% (P<0.05), respectively, while the rice yield under T3 treatment significantly decreased by 9.4% (P<0.05). Compared to CN treatment, the contents of TLOC, HLOC, and CPMI under SRN treatment significantly increased by 7.1%, 6.9% and 9.4% (P<0.05), respectively. Based on the crop rotation system and nitrogen fertilizer application methods, the rice yield, SOC, TLOC contents and CPMI under SRN treatment under CMV-R rotation system were all higher than those of the other treatments, followed by the SRN treatment under W-R rotation system and the T1 treatment under CMV-R rotation system, while no significant difference was found for these indicators between T1 treatment under CMV-R rotation system and SRN treatment under W-R rotation system. Therefore, in the rice growing areas in the middle and lower reaches of the Yangtze River, 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