Abstract: To elucidate the long-term impact of microbial insecticides on the population abundance and metabolic functional diversity of soil microorganisms in rice paddies, we employed a combination of isolation culture techniques and the Biolog microplate method to carry out comparative studies on the diversity of soil microorganisms with long-term application of the microbial insecticide CnmeGV·Bt and chemical insecticides, analyze the microbial population dynamics, metabolic functional activity, and carbon source utilization diversity. The results indicated that, compared to conventional chemical insecticides, the application of CnmeGV·Bt significantly increased the populations of bacteria, fungi, and actinomycetes by 1.86, 1.75, and 1.34 times, respectively. Further analysis revealed that the abundance of Trichodermaspp. in fungi was significantly higher under CnmeGV·Bt treatment than chemical insecticides’, while the populations of Bacillusspp., phosphate-solubilizing and potassium-solubilizing bacteria showed no significant differences. The AWCD (Average Well Color Development) values of soil microbial communities exhibited similar trends over time across treatments, with minimal changes in the first 48 hours, followed by a rapid increase between 48 and 144 hours. Notably, AWCD values were significantly higher in the CnmeGV·Bt-treated soil from 48 to 192 hours than chemical insecticides treatment. Shannon diversity index of soil microbial communities showed no significant differences between treatments, whereas Simpson and inverse Simpson index were significantly elevated under CnmeGV·Bt treatment. The microbial utilization of various carbon sources shifted following CnmeGV·Bt application, with significantly higher utilization intensities observed for seven carbohydrates, two amino acids, six hexose acids, seven carboxylic acids, esters, and fatty acids than chemical insecticides. Conversely, four carbohydrates and three carboxylic acids, esters, and fatty acids were utilized less intensively. Principal component analysis identified specific carbon sources associated with CnmeGV·Bt treatment, including glucuronamide, mucic acid, pectin, L-rhamnose, and β-methyl-D-glucoside, while chemical insecticides treatment were linked to gentiobiose, L-fucose, and D-malic acid. These findings demonstrate that replacing chemical insecticides with microbial insecticides in rice paddies can enhance soil microbial abundance and metabolic function, providing critical insights for evaluating the environmental behavior and ecological effects of Bt-based biopesticides.