AbstractIsotopic radiocarbon (Δ14C) signatures of ecosystem respiration (Reco) can identify old soil carbon (C) loss and serve as an early indicator of permafrost destabilization in a warming climate. Warming also stimulates plant productivity causing plant respiration to dominate Reco Δ14C signatures and potentially obscuring old soil C loss. Here, we investigate how a wide spatio‐temporal gradient of permafrost thaw and plant productivity affects Reco Δ14C patterns and isotopic partitioning. Spatial gradients came from a warming experiment with doubling thaw depth and variable biomass, and a vegetation removal manipulation to eliminate plant contributions. We sampled in August and September to capture transitions from high to low plant productivity, decreased surface soil temperature, and relatively small seasonal thaw extensions. We found that surface processes dominate spatial variation in old soil C loss and a process‐based partitioning approach was crucial for constraining old soil C loss. Resampling the same plots in different times of the year revealed that old soil C losses tripled with cooling surface temperature, and the largest old soil C losses were detected when the organic‐to‐mineral soil horizons thawed (∼50–60 cm). We suggest that the measured increase in old soil respiration over the season and when the organic‐to‐mineral horizon thawed, may be explained by mobilization of nitrogen that stimulates microbial decomposition at depth. Our results suggest that soil C in the organic to mineral horizon may be an important source of soil C loss as the entire Arctic region warms and could lead to nonlinearities in projected permafrost climate feedbacks.