Mesocosm experiments in the Peruvian Upwelling during a coastal El Niño examined silicon cycling and isotope dynamics under changing nutrient conditions. The addition of nitrate-depleted deep water shifted the phytoplankton community from diatoms to silicoflagellates, altering Si isotope signatures. We provide the first field-based estimate of the Si isotope fractionation factor for silicoflagellates, showing their potential importance for interpreting modern and past marine silicon cycling.