Soil freeze-thaw cycles affect N2O fluxes in high- and mid-latitude regions, but understanding microbial processes behind N2O will help clarify the long-term impact of freeze-thaw on climate change. The aim of this study was to investigate the impacts of freeze-thaw cycles on microbial abundances and N2O emissions in a hemi-boreal drained peatland forest. The soil freeze-thaw experiment involved artificial heating to thaw the topsoil after freezing. Results showed that thawing of the 5 cm topsoil increased soil water content (SWC) and N2O emissions. Microbial analysis demonstrated that the abundance of soil prokaryotes increased with thawing. N2O emissions were negatively correlated with NH4+-N while ammonia-oxidizing archaea and bacteria, including complete ammonia oxidizers, increased their abundance. This indicates a potential nitrification pathway. The abundance of nitrite reductase genes (nirK and nirS) showed a positive correlation with N2O fluxes, while nosZ genes did not increase. The results provide an insight into the impact of soil freeze-thaw cycles on N2O fluxes and the underlying microbial processes. The dynamics of SWC during the thawing period were the most direct driver of the increase in N2O emissions. Incomplete denitrification was the dominant process for the N2O emissions during the thaw. More than 80% of produced N2O was denitrified to inert N2, as shown by high potential N2 emissions. The frequency of freeze-thaw events is expected to increase due to climate change; therefore, determining the underlying microbial processes of the N2O emissions under freeze-thaw is of great importance in predicting possible impacts of climate change in forests.