Abstract 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 µm) dominated primary production (30–95%), relying mainly on nitrate to support their growth. Surface LC omega‐3 concentrations reached peaks of 215 and 175 µg L −1 pre‐ and post‐upwelling, up to 10 times higher than previous measurements from the BUS (<5 µg L −1 ). Pre‐upwelling, non‐diatom trophic markers (18:1 n − 9, 18:4 n − 3, 18:5 n − 3) were dominant, with a switch over just two days to diatom trophic markers post‐upwelling (16:1 n − 7, 16:2 n − 4, 16:2 n − 7, 16:3 n − 4, 16:4 n − 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.