The nitrogen isotopic composition of diatom frustule‐bound organic matter (δ15NDB) 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 Chaetoceros is of particular interest because of its abundance throughout ocean basins, its shifting biogeography during glacial periods, and the ability of many species to form heavily silicified resting spores. Here we investigate how Chaetoceros resting spores (CRS) record surface nitrate conditions in their nitrogen isotopic composition, and thus impact δ15NDB records, using assemblage‐specific sedimentary δ15NDB measurements and laboratory culture experiments. We find that fossil CRS from ODP Site 1098 record δ15NDB values 1.1–7.8‰ lower than non‐CRS diatoms in sediment. CRS grown in culture yield consistent results, recording δ15NDB values 2.6–8.2‰ lower than vegetative Chaetoceros in the same cultures. Low values are attributed to assimilation of isotopically light ammonium, heavy silicification, and/or internal nitrogen allocation processes during sporulation. Applying these findings to published δ15NDB records, variable CRS relative abundance in open ocean glacial sediments does not significantly bias δ15NDB records across glacial‐interglacial cycles, despite the large δ15NDB difference observed in CRS versus non‐CRS diatoms, due to the spores' small size.