Abstract: In recent decades, mineralization of soil organic carbon has gradually become a focus due to greenhouse gas (GHG) emissions. The mineralization of soil organic carbon is mainly the decomposition of soil organic carbon under the action of microorganisms, which is an important pathway for soil organic carbon loss. Chloroform has strong sterilization power, and different microorganisms have different sensitivities to it. Furthermore, the soil microbial biomass and community composition can be changed by different fumigation durations. To explore the limiting factors of soil organic carbon mineralization in purple soil farmland, soils from plots with long-term application of pig manure treatment were selected for this laboratory incubation study. The effect of different soil microbial biomasses on soil organic carbon mineralization was investigated through varying the chloroform fumigation time and observing the changes in CO₂ emission rate under different treatments. The experiment included five treatments: fumigation for 24 h (C24), 2.5 h (C2.5), 1.5 h (C1.5), and 1 h (C1); and an unfumigated control (CK). The treatment not only changed the soil microbial biomass, but also greatly changed the soil microorganism community composition, which further verified the Regulation Gate hypothesis. The results showed that after fumigation, the soil microbial residues released microbial biomass carbon and the remaining microorganisms rapidly utilized this carbon source. Due to the availability of this new carbon source, the soil CO₂ emission rate increased rapidly. The variation trend in the soil CO₂ emission rate among different treatments was consistent. Due to the microbial residue carbon source, the CO₂ emission rate of fumigation treatment was higher than that of CK within 7 days of incubation, increasing rapidly to a maximum and then decreasing to a level comparable to the initial level. The order of the maximum values of the soil CO₂ emission rate among different treatments was C2.5>C24>C1.5>C1>CK. Compared with CK, the increases were 309.01%, 182.00%, 73.85%, and 30.45%, respectively. There were significant differences among the treatments (P<0.05). The soil CO₂ emission rate increased slowly and then decreased slowly during days 7–53 of the incubation. The average CO₂ emission rates of treatments C24, C2.5, C1.5, C1, and CK were 6.01±0.43, 5.94±0.29, 6.07±0.59, 5.78±0.49, and 6.23±0.13 μg∙g⁻¹∙h⁻¹, respectively. After 32 d, the rates of fumigation treatments were slightly lower than that of CK with no significant differences among different treatments. The variation in cumulative CO₂ emissions under different treatments conformed to the model y=at^b. The higher the maximum emission rate, the smaller the value of a and the larger the value of b. The b value of all treatments was less than 1, indicating that the cumulative emission increased with incubation time with a gradually slowing rate. The results of this study support the Regulatory Gate hypothesis of soil organic carbon mineralization, which states that the mineralization of soil organic carbon is unrelated to microbial biomass size, community composition, and activity in calcareous purple soil farmland treated with pig manure over a long period of time.