The Benguela Upwelling System (BUS) is one of the world?s most productive ecosystems, supporting globally-relevant fisheries. The BUS marine community is modulated by the availability of nutrients and omega-3 long chain polyunsaturated fatty acid (hereafter, LC omega-3). Phytoplankton growth in the BUS can be supported by upwelled nitrate, a new nitrogen (N) source to the surface, or by recycled N such as ammonium. Preferential assimilation of one N source over another may yield differences in LC omega-3 production between high and low food-quality species. To evaluate how upwelling and the N source(s) consumed by phytoplankton influence LC omega-3 production, we sampled a BUS anchor station daily for 10 days. Upwelling on days 5-7 supplied high concentrations of nutrients to the surface, while pre- and post-upwelling, surface waters were stratified and nutrient concentrations were low. LC omega-3 and phytoplankton concentrations were near-zero during upwelling, and elevated pre- and post-upwelling. Throughout our sampling, nanoplankton (2.7-10?m) dominated primary production (30-95%), relying mainly on nitrate to support their growth. Surface LC omega-3 concentrations reached peaks of 215 and 175 ?gL-1 pre- and post-upwelling, up to 10-times higher than previous measurements from the BUS (<5 ?gL-1). Pre-upwelling, non-diatom trophic markers (18:1n-9,18:4n-3,18:5n-3) were dominant, with a switch over just two days to diatom trophic markers post-upwelling (16:1n-7,16:2n-4,16:2n-7,16:3n-4,16:4n-1). This study reveals the key role of upwelling in promoting phytoplankton LC omega-3 production, which is tightly coupled to the supply of new nitrate. Additionally, the high observed LC omega-3 concentrations suggest that global LC omega-3 production is underestimated. (2023-07-17)