Scientists must embrace the necessity to offset global CO2 emissions regardless of politics. Efforts to enhance terrestrial organic carbon sequestration have traditionally focused on aboveground biomass and surface soils. An unexplored potential exists in thick lower horizons of widespread, mature soils such as Alfisols, Ultisols, and Oxisols. We present a case study of fate and transport of dissolved organic carbon (DOC) in a highly weathered Ultisol, involving spatial scales from the laboratory to the landscape. Our objectives were to interpret processes observed at various scales and provide an improved understanding of coupled hydrogeochemical mechanisms that control DOC mobility and sequestration in deep subsoils within humid climatic regimes. Our approach is multiscale, using laboratory-scale batch and soil columns (0.2 by 1.0 m), an in situ pedon (2 by 2 by 3 m), a well-instrumented subsurface facility on a subwatershed (0.47 ha), and ephemeral and perennial stream discharge at the landscape scale (38.4 ha). Laboratory-scale experiments confirmed that lower horizons have the propensity to accumulate DOC, but that preferential fracture flow tends to limit sequestration. Intermediate-scale experiments demonstrated the beneficial effects of C diffusion into soil micropores. Field- and landscape-scale studies demonstrated coupled hydrological, geochemical, and microbiological mechanisms that limit DOC sequestration, and their sensitivity to local environmental conditions. Our results suggest a multi-scale approach is necessary to assess the propensity of deep subsoils to sequester organic C in situ. By unraveling fundamental organic C sequestration mechanisms, we improve the conceptual and quantitative understanding needed to predict and alter organic C budgets in soil systems.