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Abstract The Southern Ocean is a high‐nutrient, low‐chlorophyll (HNLC) region characterized by incomplete nitrate (NO₃⁻) consumption by phytoplankton in surface waters. During this incomplete consumption, phytoplankton preferentially assimilate the¹⁴N‐ versus the¹⁵N‐bearing form of NO₃⁻, quantified as the NO₃⁻assimilation isotope effect (¹⁵ε). Previous summertime estimates of the¹⁵ε from HNLC regions range from 4 to 11‰. While culture work has shown that the¹⁵ε varies among phytoplankton species, as well as with light and iron stress, we lack a systematic understanding of how and why the¹⁵ε varies in the field. Here we estimate the¹⁵ε from water‐column profile and surface‐water samples collected in the Indian sector of the Southern Ocean—the first leg of the Antarctic Circumnavigation Expedition (December 2016–January 2017) and the Crossroads transect (April 2016). Consistent with prior work in the mid‐to‐late summer Southern Ocean, we estimate a higher¹⁵ε (8.9 ± 0.6‰) for the northern Subantarctic Zone and a lower¹⁵ε (5.4 ± 0.9‰) at and south of the Subantarctic Front. We interpret our data in the context of coincident measurements of phytoplankton community composition and estimates of iron and light stress. Similar to prior work, we find a significant, negative relationship between the¹⁵ε and the average mixed‐layer photosynthetically active radiation flux of 30–100 μmol m⁻² s⁻¹, while above 100 μmol m⁻² s⁻¹,¹⁵ε increases again. In addition, while we observe no robust relationship of the¹⁵ε to iron availability or phytoplankton community, mixed‐layer nitrification over the Kerguelen Plateau appears to strongly influence its magnitude.