Both the nitrogen (N) isotopic composition (δ¹⁵N) of the nitrate source and the magnitude of isotope discrimination associated with nitrate assimilation are required to estimate the degree of past nitrate consumption from the δ¹⁵N of organic matter in Southern Ocean sediments (e.g., preserved within diatom microfossils). It has been suggested that the amplitude of isotope discrimination (i.e. the isotope effect) correlates with mixed layer depth, driven by a physiological response of phytoplankton to light availability, which introduces complexity to the interpretation of sedimentary records. However, most of the isotope effect estimates that underpin this hypothesis derive from acid-preserved water samples, from which nitrite would have been volatilized and lost during storage. Nitrite δ¹⁵N in Antarctic Zone surface waters is extremely low (−61 ± 20‰), consistent with the expression of an equilibrium isotope effect associated with nitrate–nitrite interconversion. Its loss from the combined nitrate + nitrite pool would act to raise the δ¹⁵N of nitrate, potentially yielding overestimation of the isotope effect. Here, we revisit the nitrate assimilation isotope effect in the Antarctic Zone with measurements of the δ¹⁵N and concentration of nitrate with and without nitrite, using frozen sea water samples from 5 different cruises that collectively cover all sectors of the Southern Ocean. The N isotope effect estimated using nitrate + nitrite δ¹⁵N is relatively constant (5.5 ± 0.6‰) across the Antarctic Zone, shows no relationship with mixed layer depth, and is in agreement with sediment trap δ¹⁵N measurements. Estimates of the N isotope effect derived from nitrate-only δ¹⁵N are higher and more variable (7.9 ± 1.5‰), consistent with an artifact from nitrate-nitrite isotope exchange. In the case of the Southern Ocean, we conclude that the δ¹⁵N of nitrate + nitrite better reflects the isotope effect of nitrate assimilation. The stability of this isotope effect across the Antarctic Zone simplifies the effort to reconstruct the past degree of nitrate consumption.