Changes in carbon density (i.e., carbon stock per unit area) and land cover greatly affect carbon sequestration. Previous studies have shown that land cover change detection strongly depends on spatial scale. However, the influence of the spatial resolution of land cover change information on the estimated terrestrial carbon sequestration is not known. Here, we quantified and evaluated the impact of land cover change databases at various spatial resolutions (250 m, 500 m, 1 km, 2 km, and 4 km) on the magnitude and spatial patterns of regional carbon sequestration in four counties in Georgia and Alabama using the General Ensemble biogeochemical Modeling System (GEMS). Results indicated a threshold of 1 km in the land cover change databases and in the estimated regional terrestrial carbon sequestration. Beyond this threshold, significant biases occurred in the estimation of terrestrial carbon sequestration, its interannual variability, and spatial patterns. In addition, the overriding impact of interannual climate variability on the temporal change of regional carbon sequestration was unrealistically overshadowed by the impact of land cover change beyond the threshold. The implications of these findings directly challenge current continental- to global-scale carbon modeling efforts relying on information at coarse spatial resolution without incorporating fine-scale land cover dynamics.