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The determination for properly combined application rate of organic and inorganic fertilizers is the key to coordinating soil nutrient supplies and rice growth demands, thereby obtaining improved economic and ecological returns in rice ratooning. A complete randomized block design (RCBD) trial with different configurations of organic and inorganic nitrogen fertilizers was conducted to determine the optimal substitution rate of organic nitrogen for inorganic nitrogen and its effect on the related physiological attributes and yield performance of the main and ratoon rice crops in 2018−2019. The results showed that 30% organic and 70% inorganic nitrogen in the mixture fertilizer (GM2) could produce the best effect on root activity, nitrogen nutrient uptake and its utilization, as well as the dry matter accumulation and its partitioning. This in turn resulted in improved productive panicles and harvest index, and hence increased grain yield by 10.36% and 15.48% in GM2 regime relative to that in CF treatment. Moreover, the uptake and utilization efficiency, agronomic utilization efficiency and partial productivity of nitrogen fertilizer were 44.37%, 23.82 kg/kg and 48.97 kg/kg, respectively under the optimal GM2 treatment, which were much higher than those in CF treatment by 12.66%, 5.05% and 13.4%, respectively. The results suggested that properly combined application of organic and chemical nitrogen fertilizer could improve soil nutrient supply to guarantee an efficient plant growth and rational dry matter allocation, thereby increasing the harvest index and grain yield in rice ratooning.