Nutrients, including vital organic compounds, vary in availability across ecosystems, with the potential to act as a strong source of selection for traits that increase nutrient acquisition and biosynthesis. Compared to freshwaters, marine ecosystems are much richer in the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA) and thus marine animals establishing new freshwater populations are faced with the challenge of acquiring DHA. The relative roles of the metabolic capacity and diet in the freshwater establishment process remain unresolved. We explored both metabolic and dietary responses to the nutritional constraints of freshwater environments for threespine sticklebacks (Gasterosteus aculeatus), integrating sampling of wild populations and common garden experiments in two related studies. Specifically, we examined how genetic capacity in terms of fads2 copy number and the DHA content of diets influenced phenotypes and performance in the field and lab. We collected data on: 1) relative fads2 copy numbers of wild stickleback across Europe, 2) fatty acid content of wild stickleback and their potential macroinvertebrate prey sampled in Switzerland, 3) relative availability of macroinvertebrate prey from streams with stickleback, 4) gut contents of wild Swiss stickleback, 5) fatty acid content of lab-reared stickleback reared in common garden and their chironomid diets, and 6) length, mass, and condition of lab-reared sticklebacks.