Denitrification is important in regulating soil nitrogen (N) availability and greenhouse gas emissions. Whereas, how different fertilization strategies affect soil denitrification rate and denitrifying microbial community remains contradictory. Here, four fertilization strategies including no fertilizer (F0), biostimulant: Bacillus amyloliquefaciens (BA), organic fertilizer (OF), and both of them together (BAOF) application were conducted in a pot experiment to study the changes in denitrification process and its regulation factors in acidic soils. Compared with F0, treatment with BA, OF, and BAOF significantly increased denitrification rate by 400 %, 619 %, and 331 %, respectively; increased nitrate concentration and nirS abundance, while decreased nosZ abundance. Soil nitrite reductase activity and nirK abundance were significantly increased by OF and BAOF treatments. BA and OF treatments significantly decreased nirS Chao1 index. The composition of nirS denitrifying community, but not nirK, was significantly altered by all treatments; it was mainly affect by soil pH, total N, and nitrate. The increase in soil nitrate concentration, nitrite reductase activity, and nirK abundance, as well as the reduction in nirS α diversity and nosZ abundance, jointly lead to a higher denitrification rate in fertilization treatments. The denitrifying genes explained more denitrification rate variation than soil environmental properties. These results revealed that the stimulation effect of OF on denitrification was alleviated by BA application, and nirS microbial composition was more sensitive to fertilization than nirK. Therefore, our study suggests that in future organic agricultural practices, the combination of organic fertilizer with B. amyloliquefaciens can reduce N losses caused by the soil denitrification process in acidic soils.