Phytoplankton play a crucial role in marine food webs as they supply essential fatty acids (FA) to higher trophic levels, from small pelagic fish to top predators, through the intermediary action of zooplankton. Thus, the composition and nutritional value of plankton communities expectably influence abundance and condition of predators potentially leading to spatial variation in trophic transfer. Through the analysis of the FA profile of zooplankton and European sardine (Sardina pilchardus), and of the community composition of phytoplankton and zooplankton, we investigated (i) large-scale spatial variability in the trophic transfer of FA from plankton to small pelagic fish and (ii) the factors influencing this transfer in the English Channel. We found that the FA composition of zooplankton and sardine differed between the western and eastern basins of the English Channel (WEC and EEC, respectively), reflecting differences in plankton community composition. The FA profile of sardine varied further with regard to energy allocation strategies and conditions. This suggests a strong bottom-up influence of plankton community composition on the spatial variability of FA transfer with an additional impact of fish physiological status. Understanding the reasons behind the separation pattern of sardines between the WEC and the EEC would be helpful to inform fisheries and ecosystem-based management advice.