Phytoplankton growth in the Equatorial Pacific contributes ~17% to global ocean primary production. Concentrations of nitrate in this region are elevated and phytoplankton growth is considered to be iron-limited. Here we show that phytoplankton are co-limited by iron and nitrogen over >8,000-km of the Equatorial Pacific, where experimental supply of either nutrient stimulated growth. This was driven by Prochlorococcus being stimulated by ammonium supply whereas other phytoplankton responded to iron. Consistent with this, sites in the far east of the basin—where both nitrate and ammonium were initially elevated—showed iron limitation only. Mechanistically, we find that co-limitation was regulated by the predominance of high-light (HL)-adapted HLIII and HLIV Prochlorococcus ecotypes, which lack iron-dependent enzymes required to utilize the abundant nitrate. These results have implications for how phytoplankton growth is modelled in the ocean and emergent phytoplankton community structures, with impacts on ocean biogeochemistry and food webs.