Prochlorococcus and Synechococcus account for roughly 25 % of primary production in the world’s oceans, and they play pivotal roles in the biogeochemical cycling of nutrients. This study focused on nitrogen‑source preferences of these two picocyanobacterial genera in the Kuroshio Extension (EK) (30° – 45° N), a region characterized by pronounced temperature and nutrient gradients. By combining in‑situ monitoring with laboratory culture experiments, we systematically investigated how temperature modulates the uptake and metabolic regulation of a complex nitrogen pool that included inorganic nitrogen forms (nitrate and ammonium) as well as organic nitrogen compounds (urea, amino acids). In the culture system, a ¹⁵N isotope‑labeling strategy was employed: chemically distinct nitrogen sources were mixed in equimolar ratios, enabling precise tracking of the uptake kinetics of each nitrogen form under co‑existing, multi‑nitrogen conditions. Flow cytometry was used to efficiently sort Prochlorococcus and Synechococcus cells and monitor changes in their relative abundances. The experimental results revealed that these microorganisms displayed the highest uptake velocity for ammonium and the lowest for nitrate.  Within the higher‑latitude portions of the Kuroshio, uptake of amino acids exceeded that of urea.  Moreover, at lower temperatures, the assimilation rates of glycine, glutamine, and urea increased, indicating a temperature‑dependent shift toward the utilization of these organic nitrogen moieties as alternative nitrogen. Collectively, this work provides mechanistic insights into how temperature and nitrogen form influence picocyanobacterial nitrogen acquisition and metabolism.