In recent years, due to the development and construction of commercial forests, a large scale of natural and secondary forests has been cleared and converted to monoculture plantations with simple stand structures in the subtropical regions of China. Nitrogen (N) is considered an important factor for maintaining the vegetation growth and primary productivity of forest systems, and soil microorganisms drive the key process of soil N transformation. However, the effects of subtropical forest conversion on microbial communities related to soil N transformation remain largely elusive. This paper aims to clarify the responses of microbial communities and their driving factors in the key processes of soil N transformation (N-fixation and ammonia oxidation) after subtropical forest conversion. Here, we used the real-time PCR and high-throughput sequencing technique, investigated the soil properties, function gene, community characteristics of N-fixing bacteria and ammonia oxidizers and their relationships in a secondary forest (CS) and four 5-year-old monoculture plantations, including Camellia oleifera (YC), Amygdalus persica (HT), Myrica rubra (YM), and Cunninghamia lanceolate (SM) in the Lutou National Station for Scientific Observation and Research of Forest Ecosystems in Hunan Province. The results indicated that forest conversion significantly changed the soil carbon (C), N contents, decreased the nifH gene abundance, the α-diversity of N-fixing bacteria and ammonia-oxidizing bacteria (AOB) communities, but increased the amo A gene abundances of the ammonia-oxidizing microorganisms, and the α-diversity of ammonia-oxidizing archaea (AOA) community. Moreover, forest conversion affected the composition of N-fixing bacteria and ammonia-oxidizers communities by altering the relative abundance of the dominant taxa, such as Proteobacteria, Cyanobacteria, Crenarchaeota and Thaumarchaeota. Lastly, redundancy analysis and structural equation modeling demonstrated that soil organic carbon (SOC), total nitrogen (TN), ammonium nitrogen (NH⁺₄-N) contents and pH were the key factors driving the variations in diversity and composition of soil N-fixing bacteria and ammonia-oxidizers communities. After forest conversion, reasonable fertilization was beneficial to the restoration of soil N-fixing bacteria and ammonia-oxidizers in monoculture plantation. In summary, the results provide a scientific basis for soil nutrient recovery, productivity improvement and sustainable management of monoculture plantations after subtropical forest conversion.