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In the California Current Ecosystem, upwelled water low in dissolved iron (Fe) can limit phytoplankton growth, altering the elemental stoichiometry of the particulate matter and dissolved macronutrients. Iron-limited diatoms can increase biogenic silica (bSi) content >2-fold relative to that of particulate organic carbon (C) and nitrogen (N), which has implications for carbon export efficiency given the ballasted nature of the silica-based diatom cell wall. Understanding the molecular and physiological drivers of this altered cellular stoichiometry would foster a predictive understanding of how low Fe affects diatom carbon export. In an artificial upwelling experiment, water from 96 m depth was incubated shipboard and left untreated or amended with dissolved Fe or the Fe-binding siderophore desferrioxamine-B (+DFB) to induce Fe-limitation. After 120 h, diatoms dominated the communities in all treatments and displayed hallmark signatures of Fe-limitation in the +DFB treatment, including elevated particulate Si:C and Si:N ratios. Single-cell, taxon-resolved measurements revealed no increase in bSi content during Fe-limitation despite higher transcript abundance of silicon transporters and silicanin-1. Based on these findings we posit that the observed increase in bSi relative to C and N was primarily due to reductions in C fixation and N assimilation, driven by lower transcript expression of key Fe-dependent genes.