Lack of regional measurements on nitrous oxide (N2O) flux under tile-drained conditions may cause a misunderstanding of key factors controlling N2O emissions at landscape scale. The aims of this study were to assess the spatial variations in N2O emissions and identify the regulatory effects of soil physico-chemical properties across a tile-drained agricultural landscape. N2O fluxes from winter wheat fields were measured at seventeen sites across a 30-km2 region using manual chambers during the fertilization periods (between February and May) in 2009 and 2010. All sites received nitrogen (N) fertilizer at 170–195 kg N ha−1 and the N regimes were mostly independent. Soil properties were determined in air-dried samples. The potential denitrification rates and the maximum fraction of N2O emitted through denitrification were measured in a laboratory incubation experiment in May 2009 using soils at nine sites with acetylene blockage technique. Results showed a non-significant difference in the potential denitrification between soils (p = 0.078) but a significant difference in the fraction of N2O production through denitrification (p = 0.024). Neither of these biological parameters that represented the activity of denitrifying enzymes related to soil properties and N2O emissions. Cumulative N2O emissions had significant spatial variations in 2009 (p = 0.001) but not in 2010 (p = 0.222). Topography did not affect N2O emissions mostly due to the topographic effects on soil hydrology being partly offset by tile drainage. Soil texture (clay or silt content), pH and exchangeable magnesium (Mg) related significantly (p < 0.05) to N2O emission factors (uncorrected for background emissions). We suggest that (1) soil clay content decreased gas diffusivity and promoted N2O reduction thereby controlling N2O emissions across the region, and (2) the effects of soil pH and Mg on N2O emissions indirectly reflected the effect of soil texture due to the interactions of soil properties.