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Vitamins B2, B6, B12, and folate are essential for methylation reactions and possibly influence the transport of polyunsaturated fatty acids in plasma and red blood cells (RBC). Associations between B-vitamin biomarkers and fatty acid (FA) profile were analyzed in Brazilian children and adolescents. This cross-sectional study included 249 children and adolescents, aged 9–13 years old. Dietary intake was assessed by the food frequency questionnaire and the healthy eating index (HEI). Biomarkers for vitamins B2, B6, B12, and folate were measured in plasma. The FA profile and the metabolites of one-carbon metabolism were measured in RBC. Associations were tested with multiple linear regression models. An increase of 1 nmol/L in vitamin B2 was associated with an increase of 0.19 mg/dL of EPA, 0.20 mg/dL of ARA, and 0.25 mg/dL of DHA in RBC. An increase of 1 ng/mL in plasma folate was associated with an increase of 0.14 mg/dL of EPA, 0.22 mg/dL of ARA, and 0.21 mg/dL of DHA in RBC. These findings highlight the importance of an adequate intake of vitamin B2 and folate in childhood, since they may improve the FA profile in RBCs and may help prevent cardiovascular disease.