Understanding the relationship between soil organic carbon (SOC) storage and nitrous oxide (N2O) fluxes is crucial for sustainable land management. In this study, we examined this relationship in different cropping systems, including annual cropping systems with a legacy of varying durations (3 versus 6 years) of grasslands and continuous grassland. The aim was to investigate (1) the effect of contrasting management practices on SOC storage and N2O emissions under field conditions and (2) to elucidate the drivers of N2O emissions under controlled conditions. Our findings showed that despite the influence of management practices on SOC stocks, no trade-off was observed between SOC storage at two different depths (10 and 30 cm) and N2O emissions. Furthermore, we found that SOC chemical signature, soil physical characteristics, and denitrifier activity, rather than SOC content and denitrifier abundances, were the primary factors determining N2O emission potential. It is interesting to note that although functional gene abundances were related to denitrifier activities, they did not show a direct correlation with N2O emissions. This suggests that environmental factors and microbial activity play a more substantial role in determining N2O emissions than denitrifier abundances. The inclusion of temporary grassland in a crop rotation had a positive impact on SOC stocks, either by improving or maintaining their levels. However, as a trade-off the introduction of temporary grassland also led to higher N2O emissions. These increased emissions were not directly associated with the amount of SOC stored in soil but rather with changes in soil physical and chemical conditions. Our research emphasizes the importance of understanding the complex relationships among soil characteristics, management techniques, and N2O emissions to develop practical mitigation plans for building more sustainable agricultural systems.