The Sargasso Sea is characterized by strong summertime stratification that is thought to drive oligotrophy, but export production is surprisingly similar to that of high-latitude regions with ample major nutrient supply. Here we use the summer-to-fall progression in the northwestern Sargasso Sea to investigate the relationship between upper ocean stratification and phytoplankton nitrogen (N) uptake. Euphotic zone particles collected in July, October, and December were sorted by flow cytometry, and the 15N/14N of separated prokaryotic and eukaryotic phytoplankton was analyzed. The 15N/14N of Prochlorococcus and Synechococcus was always low, indicating uniform reliance on recycled N. In July and in two fall profiles, the 15N/14N of eukaryotic phytoplankton was high, reflecting consumption of subsurface nitrate. In three other fall profiles, eukaryotic 15N/14N was similar to prokaryote 15N/14N, suggesting a shift toward more complete reliance on recycled N. The progressive deepening of the mixed layer from summer to fall, although reducing the surface-to-deep density contrast, increases the density difference of the euphotic zone as a whole from underlying nutrient-rich waters, which may play a role in the observed decline in euphotic zone nitrate supply into the fall. The apparent summertime nitrate supply to the euphotic zone, when the mixed layer is shallowest, may help to explain the surprisingly high export production of the subtropical and tropical ocean.