Northern high-latitude rivers are major conduits of carbon from land to coastal seas and the Arctic Ocean. Arctic warming is promoting terrestrial permafrost thaw and shifting hydrologic flowpaths, leading to fluvial mobilization of ancient carbon stores. Here we describe 14 C and 13 C characteristics of dissolved organic carbon from fluvial networks across the Kolyma River Basin (Siberia), and isotopic changes during bioincubation experiments. Microbial communities utilized ancient carbon (11,300 to >50,000 14 C years) in permafrost thaw waters and millennial-aged carbon (up to 10,000 14 C years) across headwater streams. Microbial demand was supported by progressively younger (14 C-enriched) carbon downstream through the network, with predominantly modern carbon pools subsidizing microorganisms in large rivers and main-stem waters. Permafrost acts as a significant and preferentially degradable source of bioavailable carbon in Arctic freshwaters, which is likely to increase as permafrost thaw intensifies causing positive climate feedbacks in response to on-going climate change.