Plasma free fatty acid (FFA) concentrations are increased in states of insulin resistance and impair endothelial function. Because the underlying mechanisms are largely unknown, we examined selected, purified FFAs' (100–300 µmol/l, 24–48 h) action on apoptosis, cell cycle distribution, and associated gene/protein expression in human umbilical vein endothelial cells (HUVECs). Stearic acid, but not oleic acid, time and concentration dependently increased endothelial apoptosis by fivefold (n=6, P<0.01), whereas polyunsaturated FFAs (PUFAs; linoleic, γ‐linolenic, and arachidonic acid) exerted proapoptotic activity only at 300 µmol/l (P<0.05). Proapoptotic FFA action increased with FFAs' number of double bonds and with protein expression of the apoptosis promotor bak. The G₀/G₁ cell cycle arrest (n=6, P<0.05) induced by stearic acid (+14%) and PUFAs (+30%) is reflected by up‐regulation of p21^WAF‐1/Cip1. In addition, all FFAs concentration dependently reduced (P<0.05) gene/protein expression of clusterin (–54%), NF‐κB's inhibitor, IκBα (–50%), endothelin‐1 (–44%), and endothelial NO synthase (–44%). Plasma samples obtained from individuals with elevated plasma FFAs (372±22 µmol/l) increased endothelial apoptosis by 4.2‐fold (P<0.001, n=10) compared with intraindividually matched low plasma FFA (56±21 µmol/l) conditions, underlining the results obtained by defined FFA stimulation. In conclusion, FFA structure differently affects endothelial cell proliferation and apoptosis, both representing key factors in the development of micro‐ and macrovascular dysfunction.