AbstractHypoxia is common feature of eutrophic estuaries and semi-enclosed seas globally. One of the key factors driving hypoxia is nitrogen pollution. To gain more insight into the effects of hypoxia on estuarine nitrogen cycling, we measured potential nitrate reduction rates at different salinities and levels of hypoxia in a eutrophic temperate microtidal estuary, the Neuse River Estuary, North Carolina, USA. We also tested the effect of hydrogen sulfide and ferrous iron additions on the nitrate reduction pathways. Overall, DNRA dominated over denitrification in this periodically hypoxic estuary and there was no correlation between the potential nitrate reduction rates, salinity, or dissolved oxygen. However, when hypoxia lasted several months, denitrification capacity was almost completely lost, and nearly all nitrate added to the sediment was reduced via DNRA. Additions of hydrogen sulfide stimulated DNRA over denitrification. Additions of ferrous iron stimulated nitrate consumption; however, the end product of nitrate consumption was not clear. Interestingly, substantial nitrous oxide formation occurred in sediments that had experienced prolonged hypoxia and were amended with nitrate. Given expanding hypoxia predicted with climate change scenarios and the increasing nitrate loads to coastal systems, coastal sediments may lose their capability to mitigate nitrogen pollution due to DNRA dominating over denitrification during extended hypoxic periods.