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This study aimed to examine the association between vitamin B-6, folate and vitamin B-12 biomarkers and plasma fatty acids in European adolescents. A subsample from the Healthy Lifestyle in Europe by Nutrition in Adolescence study with valid data on B-vitamins and fatty acid blood parameters, and all the other covariates used in the analyses such as BMI, Diet Quality Index, education of the mother and physical activity assessed by a questionnaire, was selected resulting in 674 cases (43% males). B-vitamin biomarkers were measured by chromatography and immunoassay and fatty acids by enzymatic analyses. Linear mixed models elucidated the association between B-vitamins and fatty acid blood parameters (changes in fatty acid profiles according to change in 10 units of vitamin B biomarkers). DHA, EPA) and n-3 fatty acids showed positive associations with B-vitamin biomarkers, mainly with those corresponding to folate and vitamin B12. Contrarily, negative associations were found with n-6: n-3 ratio, trans-fatty acids and oleic: stearic ratio. With total homocysteine (tHcy), all the associations found with these parameters were opposite (for instance, an increase of 10 nmol/l in red blood cell folate or holotranscobalamin in females produces an increase of 15.85 mu mol/l of EPA (P value < 0.01), whereas an increase of 10 nmol/l of tHcy in males produces a decrease of 2.06 mu mol/l of DHA (P value < 0.05). Positive associations between B-vitamins and specific fatty acids might suggest underlying mechanisms between B-vitamins and CVD and it is worth the attention of public health policies.