Description: Nitrous oxide (N2O) is a strong greenhouse gas and an ozone depleting agent. In marine environments, N2O is produced biologically via ammonium oxidation, nitrite, and nitrate reduction. The relative importance of these principle production pathways is strongly influenced by oxygen availability. We conducted 15N tracer experiments of N2O production in parallel with measurements of N2O concentration and natural abundance isotopes/isotopomers in Saanich Inlet, a seasonally anoxic fjord, to investigate how temporal and vertical oxygen gradients regulate N2O production pathways and rates. In April, June, and August 2018, the depth of the oxic‐anoxic interface (dissolved oxygen = 2.5 μmol L−1 isoline) progressively deepened from 110 to 160 m. Within the oxygenated and suboxic water column, N2O supersaturation coincided with peak ammonium oxidation activity. Conditions in the anoxic deep water were potentially favorable to N2O production from nitrate and nitrite reduction, but N2O undersaturation was observed indicating that N2O consumption exceeded rates of production. In October, tidal mixing introduced oxygenated water from outside the inlet, displacing the suboxic and anoxic deep water. This oxygenation event stimulated N2O production from ammonium oxidation and increased water column N2O supersaturation while inhibiting nitrate and nitrite reduction to N2O. Results from 15N tracer incubation experiments and natural abundance isotopomer measurements both implicated ammonium oxidation as the dominant N2O production pathway in Saanich Inlet, fueled by high ammonium fluxes (0.6–3.5 nmol m−2 s−1) from the anoxic depths. Partial denitrification contributed little to water column N2O production because of low availability of nitrate and nitrite.