AbstractSoil carbon decomposition is primarily driven by microbial activities and is regulated by factors which stimulate or impede microbial functions. Deep podzolized carbon (DPC), found in the United States Southeastern Coastal Plain, is situated well below the soil surface in horizons isolated from active plant input. This carbon is characterized by high C:N ratios (>30) which could reflect nutrient limitation of microbial decomposition. To uncover the energy or nutrient limitation on DPC degradation, a 90‐day priming experiment was performed with soils from the surface horizon and DPC horizons (i.e., Bh1 and Bh2) received the additions of 13C‐labeled alanine and glucose. This resulted in prominent priming effects: addition of alanine increased basal decomposition of soil organic carbon by 918 ± 51% and 737 ± 7% in Bh2 and Bh1, respectively. Glucose relative priming was 505 ± 28% in Bh1 and 606 ± 77% of basal respiration in Bh2. These strong responses to substrate input highlight the susceptibility of DPC to loss when microbial carbon and nutrient constraints are alleviated. After 90 days, glucose addition increased the microbial biomass in DPC horizons relative to alanine addition, with the latter showing no difference from ultrapure‐water control. The response of the microbial biomass indicates constraint by a lack of energy sources both by the paucity of labile substrates and reduced availability of organic matter as a result of podzolization. Our study has important implications for predicting the response of DPC in Coastal Plain soils in the context of land management and global change.