Two meridional transects were conducted in the tropical and subtropical Atlantic to describe (i) the spatial variability of total and size-fractionated (picophytoplankton and phytoplankton > 2 μm) chlorophyll a (chl a) concentration and primary production, (ii) the relative contribution of each phytoplankton size fraction to total biomass and carbon fixation, and (iii) the spatial variability of size-fractionated phytoplankton growth rate (P/B) and assimilation number (P/chl a) in the ocean. The highest chl a for both size fractions was observed in the Western Tropical Atlantic province (WTRA), while the lowest chl a was found in the upper mixed layer (UML) of the South Atlantic Tropical gyre (SATL). A similar pattern was found for carbon fixation. Within the SATL, the highest picophytoplankton contribution to total production was recorded at the Deep Chlorophyll Maximum (DCM), while the contribution of phytoplankton > 2 μm was higher in the UML. Additionally, the relative contribution of large phytoplankton to total integrated primary production was higher than its contribution to total biomass. Both size fractions depicted maximum P/B and P/chl a in WTRA surface waters. In the SATL province, phytoplankton > 2 μm showed the highest P/B and P/chl a along the UML, while picophytoplankton P/B and P/chl a peaked around the DCM. We suggest that the differential impact of light on small and large phytoplankton may help to explain the contrasting dynamics of the two size classes.