Carbon (C) buried deep in soil (below 1 m) is often hundreds to thousands of years old, though the stability and sensitivity of this deep C to environmental change are not well understood. We examined the C dynamics in three soil horizons and their responses to changes in substrate availability in a coarse-textured sandy spodosol (0.0–0.1, 1.0–1.3, and 2.7–3.0 m deep). Substrate additions were intended to mimic an increase in root exudates and available inorganic nitrogen (N) that would follow an increase of belowground biomass at depth, as previously found in a long-term CO2 enrichment experiment at this site. We incubated these soils for 60 days with glucose, alanine, and leaf litter, crossed with an inorganic N amendment equivalent to three times ambient concentrations. The organic substrates were isotopically labeled (13C), allowing us to determine the source of mineralized C and assess the priming effect. Enzyme activity increased as much as 13 times in the two deeper horizons (1.0–1.3, and 2.7–3.0 m) after the addition of the organic substrates, even though the deepest horizon had microbial biomass and microbial phospholipid fatty acids below the level of detection before the experiment. The deepest horizon (2.7–3.0 m) yielded the largest priming response under alanine, indicating that microorganisms in these soil horizons can become active in response to input of organic substrates. Inorganic N amendments significantly decreased the priming effect, suggesting that decomposition may not be N limited. However, alanine (organic N) yielded the highest priming effect at every soil depth, indicating the importance of differentiating effect of organic and inorganic N on decomposition. Distinct priming effects with depth suggest that portions of the soil profile can respond differently to organic inputs. Our findings indicate that the deep soil C pools might be more vulnerable to environmental or anthropogenic change than previously thought, potentially influencing net CO2 exchange estimates between the land and the atmosphere.