15N nitrate uptake assays were conducted using tracer additions of highly enriched (98%) 15N-labeled nitrate for four Arctic-adapted strains of Thalassiosira gravida and three temperate-adapted strains of Thalassiosira rotula. The experiments were carried out to understand the nitrate uptake kinetics of these strains under controlled laboratory conditions. The conduction of the assays comprised in this dataset took place between 2022-03-09 and 2022-07-15 at the Alfred Wegener Institute, Bremerhaven, Germany. Prior to the experiment, the Arctic strains were maintained at 4°C and the temperate strains at 15°C in climate chambers, with a light intensity of 30 µmol photons m-2s-1 and a photoperiod of 16:8h light:dark. The cultures were kept in exponential growth by semi-continuous dilution with K-medium, enriched with silicon and devoid of ammonium. For precultivation, 2L batch cultures of each strain were grown in ESAW medium at a light intensity of 25 µmol photons m-2 s-1 and a light:dark cycle of 16:8h. Arctic strains were cultivated at 9°C, while temperate strains were grown at 16°C, representing their respective lowest optimum temperatures. The initial nitrate concentration in the medium was set to approximately 100 µM L-1 (equivalent to 1/5 K-medium) to ensure sufficient biomass for subsequent 15N isotope analysis. Upon reaching mid-exponential growth phase, a nitrogen depletion procedure was implemented. This involved decanting 90% of the supernatant from the batch culture flasks and resuspending the sedimented cells in nitrate- and ammonia-free modified ESAW medium. This process was repeated twice more over the course of 48 hours, allowing cells to sediment between each dilution step. This dilution series resulted in a calculated final nitrate concentration of 0.1 µM NaNO3 L-1 at maximum, not accounting for ongoing nitrogen uptake by the diatoms, which likely further depleted the dissolved nitrate concentrations. This approach was deemed the best compromise between maintaining a sufficiently dense culture and achieving nitrogen depletion. 24 hours after the final dilution with nitrogen-free medium, the uptake assay was prepared. Aliquots of 40 ml culture from the batch cultures were transferred into 50 ml culture flasks for the nitrate addition treatments. The experimental design included seven nitrate levels (each with four replicates) and a nitrate-depleted control treatment. The seven nitrate concentrations tested were 0.1, 0.4, 0.8, 2, 10, 50, and 100 µmol NaNO3 L-1. For concentrations of 2 µmol NaNO3 L-1 and below, a 1:1 ratio of 14N:15N was used, while a 9:1 ratio was employed for concentrations above 2 µmol NaNO3 L-1. The experimental units were incubated for 40 minutes under the same conditions as the batch cultures. Following incubation, the samples were filtered onto pre-combusted glass microfiber filters (Whatman GF/F, Maidstone, UK). To reduce dissolved 15N contamination on the filter, an additional 100 ml of nitrate-free medium was filtered through each sample. The filters were then dried at 60°C for 48 hours before being folded into tin capsules and stored in the dark until further analysis. Particulate organic carbon (POC), particulate organic nitrogen (PON), and the particulate 15N:14N ratio were measured using an elemental analyzer (Flash 2000, Thermo Scientific, Karlsruhe, Germany) coupled to an isotope ratio mass spectrometer (IRMS, Thermo Scientific, Karlsruhe, Germany), following the methodology described by Morrien et al. (2017).