At least 1218 Pg (billion tons) of soil carbon (C) are stored in permafrost soils in boreal and arctic ecosystems, almost twice as much C than currently contained in the atmosphere. Permafrost thaw, and the microbial decomposition of previously frozen organic C, is considered one of the most likely positive feedbacks from terrestrial ecosystems to the atmosphere in a warmer world. Yet, the rate of release is highly uncertain but crucial for predicting the strength and timing of this C cycle feedback, and thus how important permafrost thaw will be for climate change this century and beyond. We report results from a tundra landscape undergoing permafrost thaw, where net ecosystem C exchange and the radiocarbon age of ecosystem respiration were measured to determine the influence of old C loss on ecosystem C balance. Sustained transfers of C to the atmosphere that could cause a significant positive feedback to climate change must come from old C, which forms the bulk of the permafrost C pool that accumulated over thousands of years. Results/Conclusions By partitioning respiration sources, we determined that areas that thawed over the past 15 years had 40% more annual losses of old C compared to minimally thawed areas, but had overall net ecosystem C uptake as increased plant growth offset these losses. In contrast, areas that thawed decades earlier lost even more old C, a 78% increase over minimally thawed areas, which contributed to overall net ecosystem C release despite increased plant growth. These data document significant losses of soil C with permafrost thaw that, over decadal time scales, overwhelms increased plant C uptake at rates that could make permafrost a large biospheric C source in a warmer world, similar in magnitude to current C fluxes from land use change.