Despite the importance of nitrous oxide (N2O) in the global radiative balance and atmospheric ozone chemistry, its sources and sinks within the Earth's system are still poorly understood. In the ocean, N2O is produced by microbiological processes such as nitrification and partial denitrification, which account for about a third of global emissions. Conversely, complete denitrification (the dissimilative reduction of N2O to N2) under suboxic/anoxic conditions is the only known pathway accountable for N2O consumption in the ocean. In this work, it is demonstrated that the biological assimilation of N2O could be a significant pathway capable of directly transforming this gas into particulate organic nitrogen (PON). N2O is shown to be biologically fixed within the subtropical and tropical waters of the eastern South Pacific Ocean, under a wide range of oceanographic conditions and at rates ranging from 2 pmol N L-1 d- to 14.8 nmol N L-1 d-1 (mean ± SE of 0.522±1.06 nmol N L-1 d-1, n = 93). Additional assays revealed that cultured cyanobacterial strains of Trichodesmium (H-9 and IMS 101), and Crocosphaera (W-8501) have the capacity to directly fix N2O under laboratory conditions; suggesting that marine photoautotrophic diazotrophs could be using N2O as a substrate. This metabolic capacity however was absent in Synechococcus (RCC 1029). The findings presented here indicate that assimilative N2O fixation takes place under extreme environmental conditions (i.e., light, nutrient, oxygen) where both autotrophic (including cyanobacteria) and heterotrophic microbes appear to be involved. This process could provide a globally significant sink for atmospheric N2O which in turn affects the oceanic N2O inventory and may also represent a yet unexplored global oceanic source of fixed N.