Field tests were conducted at experimental station of Chongluo, Nanping city, Fujian province, China in 2018–2019 to explore the action and physiological mechanism of organic nitrogen in rice ratooning under the configurations with different proportions of organic and inorganic nitrogen. The results showed that sole chemical fertilizer (CF) led to a significant decrease in soil nutrient availability, especially at late growth stage, and reduced grain yield of the main and ratooning crops compared with the optimal fertilizer configuration (GM2) in 30% organic nitrogen with 70% chemical nitrogen fertilizer in the premise of total application rate at 225 kg N/ha. Further studies have revealed that GM2 relative to CF treatment promoted to produce more productive panicles of the main crop and had carryover effect on the regeneration rate of ratooning rice due to national C13‐assimilates partitioning in rice–rhizosphere systems, which resulted in increased soil microbial diversity and soil fertility, thereby enhanced yields of the main and ratooning rice crops. These findings help to better understand the action and mechanism of the proper combined application of organic and inorganic nitrogen fertilizer via improving remobilization of 13C assimilates in rice–rhizosphere system, and ultimately increasing grain yield in rice ratooning.