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 conduct comparative studies on the diversity of soil microorganisms with long-term application of microbial insecticide CnmeGV·Bt and chemical insecticides, and to analyze microbial population dynamics, metabolic functional activity, and carbon source utilization diversity. The results indicated that, compared to aplicaion of conventional chemical insecticides, 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 Trichoderma spp. of fungi was significantly higher under CnmeGV·Bt treatment than under chemical insecticidal treatment, whereas the populations of Bacillus spp., phosphate and potassium solubilizing bacteria, showed no significant differences. The average well color development (AWCD) values of soil microbial communities exhibited similar trends over time across treatments, with minimal changes in the first 48 h, followed by a rapid increase between 48 and 144 h. Notably, AWCD values were significantly higher in the CnmeGV·Bt-treated soil from 48 to 192 h than those in the chemical insecticidal treatment. The Shannon diversity index of soil microbial communities showed no significant differences between treatments, whereas the Simpson and inverse Simpson indices were significantly elevated under CnmeGV·Bt treatment. Compared with chemical insecticidal application, 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. Conversely, four carbohydrates, three carboxylic acids, esters, and fatty acids were less intensively utilized. Principal component analysis identified specific carbon sources associated with CnmeGV·Bt treatment, including glucuronamide, mucic acid, pectin, L-rhamnose, and β-methyl-D-glucoside, whereas chemical insecticidal treatment was linked to gentiobiose, L-fucose, and D-malic acid. These findings demonstrate that replacing chemical insecticides with microbial insecticides in rice paddies can enhance the soil microbial abundance and metabolic function. The results provide critical insights into the environmental behavior and ecological effects of Bt-based biopesticides.