Abstract ： To determine the optimal substitution ratio of organic fertilizer for chemical nitrogen and its underlying mechanisms in rice-crayfish co-culture systems, a two-year (2023-2024) field experiment was conducted with five treatments: no nitrogen control (R), conventional chemical fertilizer (RS), and organic fertilizer substituting 30% (RS3), 50% (RS5), and 70% (RS7) of chemical nitrogen. Rice yield, nitrogen use efficiency, soil nitrogen mineralization characteristics, and humic acid structure were systematically analyzed. The results demonstrated interannual cumulative effects of organic substitution on yield and nitrogen use efficiency. The RS3 treatment maintained high yields in both years, with a significant increase in 2024 (9.12 t/hm2), and its agronomic efficiency and partial factor productivity were also significantly higher than those of the other treatments, whereas RS7 showed the lowest yield and nitrogen efficiency. Soil nitrogen mineralization parameters varied regularly with the substitution ratio. The RS3 treatment maintained the highest labile mineralizable nitrogen pool (N?, 90.04 mg/kg) and a moderate mineralization rate (k?, 0.135 day?1), resulting in a better match between soil nitrogen supply and crop demand. Although the RS7 treatment exhibited the highest slowly mineralizable nitrogen pool (N?) and total mineralizable nitrogen (N?+N?), its N? and k? were significantly lower, indicating insufficient seasonal nitrogen supply. Continuous organic substitution significantly increased soil cation exchange capacity and humic acid content. However, the humic acid in the RS7 treatment showed a numerically higher aliphatic/aromatic carbon ratio, suggesting a more aliphatic structure and weaker nitrogen retention capacity compared with RS3. In conclusion, 30% organic substitution maintained a high labile nitrogen pool and high aromaticity of humic acid, thereby balancing seasonal nitrogen supply with long-term soil fertility improvement. This ratio represents a suitable strategy for synergistically improving yield and efficiency in rice-crayfish co-culture systems.