Trans‐palmitoleic acid (trans‐9 C16:1, or trans‐C16:1 n‐7, TPA) is typically found in ruminant‐derived foods (milk and meat). Of note, previous epidemiological studies associated high levels of circulating TPA with a lower risk of type 2 diabetes and metabolic syndrome in humans. At the current time, TPA intakes in humans are ensured by ruminant‐derived foods. However, due to the very low commercial availability of TPA, there are no supplementation studies carried out so far. Therefore, the ability for dietary TPA to prevent type 2 diabetes and metabolic syndrome has never been experimentally assessed. Here, we report a method (among others) to get dozens of grams of pure TPA as ethyl ester, to perform dedicated supplementation studies. For that purpose, we started from food sources containing high amounts of cis‐palmitoleic acid (cis‐9 C16:1, or cis‐C16:1 n‐7, CPA), dealing with fatty acids ethyl esters all along with the experiment. CPA was purified with flash LC, then submitted to a cis/trans isomerization step. Finally, TPA was separated from CPA by low‐temperature crystallization in methanol. The final product was fully characterized by 1H and 13C nuclear magnetic resonance spectrometry. We were able to produce approx. 70 g of 85%‐purity TPA suitable for nutritional studies.