Nitrous oxide (N2O) emissions from soils applied with livestock excreta have been widely reported previously. The highest N2O emissions from soils are also often reported during thawing periods in cold regions where soil freezing is common. However, the combined effects of cow urine application and freeze-thaw events on N2O emissions and the related enzyme activities are still not clear. Thus, we simulated a freeze-thaw event at -3 °C for 7 days, and then increased to 3 °C for 46 days using intact soil cores with cow urine (392 kg N ha-1). We compared the factors influencing the magnitudes of N2O emissions through soil microbial processes with and without the freeze-thaw event. Dicyandiamide (DCD), an inhibitor of nitrification, was added to investigate the significance of nitrification on N2O emissions. The N2O emission rates from the urine-applied soils peaked to approximately 1000 μg N2O-N m-2 h-1 immediately after the soils thawed. Soil freezing with urine application was significantly higher cumulative N2O emissions (537 mg N2O-N m-2), compared to non-frozen soils with urine (247 mg N2O-N m-2) during the incubation period (54 days). The effect of DCD application on N2O emissions was not clear during the freeze-thaw event, although nitrate production rates were reduced. After the freezing event, soil moisture (water-filled pore space) was significantly higher in the non-frozen soils compared to the frozen soils, due to a 9% decline in bulk density of frozen soils. Additionally, the impact of thawing on urease and denitrification enzyme activities was influenced by the urine application. Urine application increased the urease activity, while the freezing event decreased the magnitudes. The physical changes in the soils were also important controlling factors of the N2O emissions from cow urine-applied soils in cold regions.