Porpose <p>Autotrophic microorganisms, especially in paddy soil, have substantial carbon (C) fixation potential, which is strongly influenced by nutrient management practices in field. However, the impacts of different fertilizer applications on microbial CO₂ fixation potential in paddy fields are not clearly addressed. Therefore, this study aims to investigate the impacts of paddy soil C fixation dynamics and the mechanisms driven by autotrophic microorganisms under different long-term fertilization regimes.</p> Materials and methods <p>Soil from a lasted 30 years of fertilized paddy fields was used, consisting of the four treatments: control (CK), mineral fertilizer (NPK), a combination of rice straw and mineral fertilizer (SNPK), and a combination of 30% manure with 70% mineral fertilizer (MNPK). After 28 days of continuous labeling with 5% (v/v) ¹³C-CO₂, the ¹³C-labeled organic carbon (¹³C-SOC), gene abundances (<i>cbbL</i>, <i>cbbM</i> and <i>accA</i>) and the ¹³C-PLFA population structures of each treatment soils have been analyzed.</p> Results and discussion <p>The results showed that the SNPK treatment significantly enhanced paddy soil CO₂ fixation capacity. The changes in CO₂-fixation capacity were more greatly attributed to the shifts in microbial community composition rather than alterations in the abundance of functional autotrophic microorganisms. Notably, random forest analysis shows ¹³C-PLFA content of actinomycetes was the key factor influencing the amount of ¹³C-SOC assimilations, revealing that actinomycetes exhibited higher carbon fixation efficiency compared to other microbial counterparts. Moreover, the significantly positive correlation between soil C/N ratio and ¹³C-SOC assimilations highlights the important role of nutrient availability in C-fixation of long-term fertilized paddy soil.</p> Conclusions <p>In conclusion, this study demonstrated that long-term fertilization could modulate paddy soil carbon fixation capacity by altering the soil C/N ratio.</p>