The microalga Nannochloropsis gaditana is a natural producer of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). TAG accumulation is induced by nitrogen starvation. The biomass-specific photon supply rate used had an effect on EPA and TAG accumulation during nitrogen starvation as well as on the localization of EPA accumulation. Clear differences in TAG yield on light were found for different biomass-specific photon supply rates and light regimes during nitrogen starvation. De novo EPA synthesis or the translocation of EPA between lipid fractions might be limiting for EPA accumulation in TAG. Further studies are needed to fully understand EPA accumulation in TAG during nitrogen starvation. To elucidate the function of EPA in TAG nitrogen recovery, experiments are suggested. The overexpression of genes involved in de novo EPA synthesis and translocation is proposed to elucidate the exact metabolic routes involved in these processes during nitrogen starvation. This work addresses future opportunities to increase EPA accumulation.