The nitrogen isotopic composition of diatom frustule-bound organic matter (δ<sup>15</sup>N<sub>DB</sub>) is often used to study changes in high latitude biological pump efficiency across glacial-interglacial cycles, but the proxy may be biased by species-specific effects. The genus <em>Chaetoceros</em> is of particular interest because of its abundance throughout ocean basins, its shifting biogeography during glacial periods, and many species’ ability to form heavily silicified resting spores. Here we investigate how <em>Chaetoceros</em> resting spores (CRS) record surface nitrate conditions in their nitrogen isotopic composition using both laboratory culture experiments and assemblage-specific sedimentary δ<sup>15</sup>N<sub>DB</sub> measurements. We find that CRS record δ<sup>15</sup>N<sub>DB</sub> values 7.0 ± 2.6 ‰ lower than vegetative <em>Chaetoceros</em> in culture and 5.6 ± 1.9 ‰ lower than non-CRS diatoms in sediment. Low values are attributed to assimilation of isotopically light ammonium, heavy silicification, and internal fractionation during sporulation. Despite the large δ<sup>15</sup>N<sub>DB</sub> difference observed in CRS versus non-CRS diatoms, increased CRS relative abundance in open ocean glacial sediments does not significantly bias δ<sup>15</sup>N<sub>DB</sub> records due to the spores’ small size.