Anorexia nervosa (AN) is a psychiatric disorder affected by psychological, environmental, and biological factors. Individuals with AN avoid high-fat, high-calorie diets and have shown abnormal metabolism of fatty acids (FAs), which are essential for brain and cognitive/neuropsychiatric health. To clarify the relationship between FAs and AN, fasting and postprandial plasma FAs in AN patients and age-matched control women were analyzed via mass-spectrometry. Clinical phenotypes were assessed using Becker Anxiety Inventory and Becker Depression Inventory. AN patients and controls exhibited different FA signatures at both fasting and postprandial timepoints. Lauric acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and alpha-linoleic acid (ALA) were higher in AN than in controls (lauric acid: 15,081.6 ± 14,970.2 vs. 8257.4 ± 4740.2 pmol/mL; ALA at fasting: 2217.7 ± 1587.6 vs. 1087.9 ± 821.2 pmol/mL; ALA at postprandial: 1830.9 ± 1115.6 vs. 1159.4 ± 664.7 pmol/mL. EPA: 33,788.3 ± 17,487.5 vs. 22,860.6 ± 12,642.4 pmol/mL; DPA: 32,664.8 ± 16,215.0 vs. 20,969.0 ± 12,350.0 pmol/mL. FDR-adjusted -values < 0.05). Food intake and AN status modified the correlations of FAs with body mass index (BMI), depression, and anxiety. Desaturases SCD-18 and D6D showed lower activities in AN compared to controls. Altered FA signature, specifically correlations between elevated -3 FAs and worsened symptoms, illustrate metabolic underpinnings in AN. Future studies should investigate the mechanisms by which FA dysregulation, specifically elevated -3 FAs, affects AN risk and outcome.