The objective of this study was to examine the relationship between amino acid (AA) intake and serum lipid profile in European adolescents from eight European cities participating in the cross-sectional (2006-2007) HELENA (Healthy Lifestyle in Europe by Nutrition in Adolescence) study, and to assess whether this association was independent of total fat intake. Diet, skinfold thickness, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), TC/HDL-c ratio, low-density lipoprotein cholesterol (LDL-c), apolipoprotein B (Apo B), apolipoprotein A1 (Apo A1) and Apo B/Apo A1 ratio were measured in 454 12.5- to 17.5-year-old adolescents (44% boys). Intake was assessed via two non-consecutive 24-h dietary recalls. Data on maternal education and sedentary behaviors were obtained via questionnaires. Physical activity was objectively measured by accelerometry. Alanine, arginine, asparaginic acid, glycine, histidine, lysine and serine intakes were inversely associated with serum TG concentrations in both boys and girls. Intake of other AA like alanine and/or arginine was also inversely associated with serum TC, LDL-c and Apo B/Apo A1 ratio only in girls. An inverse association was observed between intakes of alanine, isoleucine, leucine, methionine, serine, tryptophan, tyrosine and valine and TC/HDL-c ratio among female adolescents. Similar results were found in males for serine and tryptophan intakes. It is noteworthy, however, that associations were no longer significant in both genders when total fat intake was considered as a confounding factor. In this sample of adolescents, the association between AA intakes and serum lipid profile did not persist when dietary fat was considered. Therefore, dietary interventions and health promotion activities should focus on fat intake to improve lipid profile and potentially prevent cardiovascular disease.