Anthropogenic sources of nitrous oxide (N₂O) have received a great deal of attention in recent years. Agricultural fertilization has been shown to be one of the most important sources of N₂O. However, the impact of urbanization in the agricultural watershed on regional N₂O emission is currently not well understood. China has been undergoing rapid urbanization, which has had a stimulating effect on aquatic N₂O emissions. Accordingly, there is an urgent need for assessment of the regional N₂O budget. Therefore, this study was conducted to reveal the N₂O emission characteristics and possible response mechanisms of a typical headstream under the human disturbed landscape gradients. The specific objectives were to investigate how nitrogen is transported and transformed with landscape gradient, as well as whether N₂O emission fluxes are significant and responsive to the load gradient over spatial and temporal scales in the South Tiaoxi River (STR), which is the most important headwater stream in the Taihu Lake Basin. N₂O emission rates ranged from -18.11 to 397.42 μg·m^-2·h^-1 and averaged (30.37 ± 10.87) μg·m^-2·h^-1. Moreover, the emissions followed a seasonal pattern, with the lowest values (9.69 ± 7.10) μg·m^-2·h^-1 being observed in winter, moderate values (17.17 ± 17.35) μg·m^-2·h^-1 occurring in summer, and the highest values (125.97 ± 90.77) μg·m^-2·h^-1 being recorded during the flood season. The significantly high N₂O emission rates during the flood period were mainly ascribed to the large amount of overland runoff input caused by continuous rainfall. N₂O emission rates responded to the landscape gradient in the watershed, gradually increasing along the stream from the upper headwaters region to the lower farmland and urban areas. Statistical analyses revealed that the nitrate (NO₃^--N) load predominantly determined the variations in stream N₂O emission rates, while other factors such as phosphorus content, dissolved oxygen, and topography played lesser roles. The South Tiaoxi River was found to be a net source of N₂O to the atmosphere, with total annual N₂O emissions from the main stream of the STR estimated to be as high as 0.38 t/a. This case study demonstrated that allochthonous pollution load inputs might dramatically stimulate aquatic N₂O emissions from a montane headstream with relatively little sediment. Since the exchange of N₂O across the gas-water interface is a complex process that involves river microbial activities, pollution loading, gas-water exchange dynamic factors as well as river hydrology and hydraulics, its mechanism requires deeper and more extensive study to enable more accurate estimation of N₂O emission fluxes and relevant climate effects at the global scale.