We characterized the effect of large-scale (>20 mm) soil physical structure on the age and recalcitrance of soil organic carbon (SOC) in upper (A) and lower (B) horizons of grassland soils from California's Central Valley. The radiocarbon content of SOC from surfaces and interiors of large-scale soil structural units (“peds”) was measured in order to characterize the spatial distribution of soil C pools with distinct residence times. The radiocarbon content of CO2 released following sieving was used to identify the C that is readily respired upon physical disturbance of soil structure. We found the longest SOC residence times in the interiors of peds from subsurface B horizons, where limited bioturbation leads to stable large-scale structure. The radiocarbon value of this interior SOC (Δ14C = −555‰) indicates that this pool has been protected from decomposition for thousands of years. Similarly ancient C (Δ14C = −596‰) was released upon physical disruption of subsurface B horizons from a similar soil, indicating that this SOC was old, but chemically labile. With cultivation, the C released upon physical disruption of B horizons was even older (Δ14C = −812‰) than in the uncultivated soil. In uncultivated A horizons, which are subject to continuous bioturbation, large-scale structure resulted in contrasting SOC pools only in the surface horizon, where “bomb” C effects are strong. A horizon incubations also suggested effects of smaller-scale structure. Loss of the labile SOC that is physically protected by large-scale structure contributes to the rapid reduction in natural soil C inventories following cultivation.