The Benguela Upwelling System (BUS) is one of the world’s most productive ecosystems, supporting globally relevant pelagic fisheries. BUS marine community can change as a function of nutrients and omega-3 long chain polyunsaturated fatty acids (hereafter, omega-3) availability. Phytoplankton growth is supported by upwelled nitrate, a new source of nitrogen (N), or by recycled N forms such as ammonium. Preferential assimilation of one N form over another may lead to differences in omega-3 production between high and low food-quality species. This study evaluates how upwelling and the N source(s) used by phytoplankton influence omega-3 production. Sampling was conducted in the BUS at an anchor station sampled daily for 10 consecutive days. An upwelling event on days 5-6-7 supplied high concentrations of nutrients to surface waters, while pre- and post- upwelling the water column was well-stratified with low nutrient concentrations. Omega-3 and phytoplankton concentrations declined to ⁓zero during the upwelling event. Nanoplankton (2.7-10µm) were responsible for most of the productivity (30-95%) and relied on nitrate for their growth. Omega-3 concentrations at the surface reached peaks of 215.5 and 175.3µgL-1 pre- and post-upwelling, which were up to 10-times higher than previous measurements from the BUS. Pre-upwelling, non-diatom trophic markers were dominant, with a rapid switch (over just two days) to diatom trophic markers post-upwelling. This study defines the key role of upwelling in promoting phytoplankton omega-3 production, which is tightly coupled to the introduction of new-N during upwelling. The high concentrations of omega-3 reported suggest that global omega-3 production is largely underestimated.