Nitrous oxide (N2O), a potent greenhouse gas, is approximately 300 times more effective at trapping heat than carbon dioxide (CO2). Over 70% of global N2O emissions originate from agricultural practices, including synthetic nitrogen fertilizer application (0.6–14.8 Tg N2O-N per year) and manure use (0.6–3.1 Tg per year). This chapter explores the biological and physicochemical mechanisms of N2O emissions, with a focus on microbial nitrification, denitrification, and lesser-known pathways such as dissimilatory nitrate reduction to ammonium (DNRA) and nitrifier-denitrification. Denitrifying microbes, including bacteria and fungi, play key roles in reducing nitrates to N2O under oxygen-limited conditions. Soil pH, moisture, and agricultural inputs significantly influence microbial N2O emissions. For example, high nitrogen fertilizer rates, such as 250 kg ha−1, have been shown to accelerate denitrification and N2O production. Biochar amendments, while promising, increased N2O emissions by 1.96–4.18 times in certain studies, likely due to enhanced microbial activity. Conservation tillage strategies reduced cumulative N2O emissions by up to 73.58% compared to conventional plowing. Additionally, hotspots like flooded depressions contribute disproportionately to total emissions, accounting for 30 ± 1% of N2O budgets within managed croplands. Recent advancements in omics technologies have enhanced our understanding of microbial diversity and functional genes driving N2O emissions. This chapter underscores the need for integrated mitigation strategies, such as optimizing nitrogen use efficiency, enhancing soil carbon storage, and leveraging microbial community engineering, to address the dual challenges of global food security and climate change.