Hyperlipidemia is found to be associated with changes in fatty acid (FA) profiles. The aim of this study was to investigate the effects of AHA-Step-1 dietary treatment and combination of fibrates (gemfibrozil) with dietary intervention on serum and erythrocyte phospholipid FA composition in human hyperlipidemia. 78 study participants with hyperlipidemia were divided in two groups. In D group (n = 41) subjects followed AHA-Step-1 diet (<30% of total from fat, <10% of energy from saturated fat, and <300 mg cholesterol per day). D+F group (n = 37) followed Step-1 diet and were receiving gemfibrozil (300 mg/twice per day). Serum lipid levels and phospholipid serum and erythrocyte FA compositions were analyzed at the beginning and after 12 weeks of treatment. Alteration in serum and erythrocyte phospholipid FA profile were found in both groups. After both treatments we found significantly higher serum phospholipid percentages of n-3, n-6 and total polyunsaturated FA. Linoleic (LA, n-6) and docosahexaenoic acid (DHA, n-3) were higher in D group, but arachidonic (AA, n-6) and linolenic acid (LNA, n-3) in D+F group. In erythrocyte phospholipid levels of stearic, palmitoleic (16 : 1, n-7) and LA were significantly higher in D group, but palmitic acid, AA and eicosapentaenoic acid (EPA, n-3) in D+F group. Stronger correlation between serum triglycerides with EPA and DHA in erythrocyte membrane phospholipid was found in D+F group. Markedly increased percentage of AA in serum and erythrocyte membrane phospholipid in hyperlipidemic patients receiving gemfibrozil on Step-1 diet is especially important for physiological functions (inflammation, vascular tone, hemostasis etc.) in relation to cardiometabolic risk.