The temperature sensitivity of ecosystem respiration regulates how the terrestrial carbon sink responds to a warming climate but has been difficult to constrain beyond the plot scale. We use observations of atmospheric CO₂ concentrations from a network of towers together with carbon flux estimates from terrestrial biosphere models to characterize the temperature sensitivity of ecosystem respiration, as represented by the Arrhenius activation energy, over various North American biomes. We infer activation energies for major biomes, which are substantially below those reported for plot-scale studies. This discrepancy suggests that sparse plot-scale observations do not capture the spatial-scale dependence and biome specificity of the temperature sensitivity. We further show that adjusting the apparent temperature sensitivity in model estimates markedly improves their ability to represent observed atmospheric CO₂ variability. This study provides observationally constrained estimates of the temperature sensitivity of ecosystem respiration directly at the biome scale and reveals that temperature sensitivities at this scale are lower than those based on earlier plot-scale studies. These findings call for additional work to assess the resilience of large-scale carbon sinks to warming.