ABSTRACT Background Although epidemiological studies suggest a protective role of B vitamins and omega-3 (ω-3) fatty acids in cognitive decline, findings from intervention studies are conflicting. Mechanistic studies suggest that the ω-3 (n–3) fatty acid status can modulate the effects of B vitamins on cognitive decline. Objectives We investigated the interaction between baseline ω-3 fatty acid statuses and folic acid treatment on cognitive decline in a placebo-controlled trial [FACIT (Folic Acid and Carotid Intima-media Thickness)]. Methods This post hoc analysis included 791 older adults aged 50–70 y with plasma total homocysteine ≥13 µmol/L and ≤26 µmol/L and serum vitamin B12 ≥200 pmol/L. Participants received 800 µg folic acid or placebo daily for 3 y. Global cognitive functioning and domain-specific functioning (episodic memory, information processing speed, executive functioning) were assessed at baseline and after 3 y. The effect of the folic acid supplementation was analyzed according to tertiles of baseline ω-3 fatty acid concentrations using linear multiple regression. Results The mean ± SD age of the study population was 60.2 ± 5.6 y, and the mean ± SD Mini-Mental State Examination score was 28.6 ± 1.5. The treatment effect of folic acid was significantly larger in participants in the low compared to high ω-3 fatty acid tertile for global cognition (difference in z-score: mean ± SE = 0.16 ± 0.059; P < 0.01). Regarding domain-specific functioning, similar results were observed for information processing speed (mean ± SE = 0.167 ± 0.068; P = 0.01). There were no overall interactions between folic acid treatment and ω-3 fatty acid tertiles for episodic memory (P = 0.14) and executive functioning (P = 0.21). Conclusions This post hoc analysis revealed that the efficacy of folic acid treatment on cognitive functioning is dependent on the ω-3 fatty acid status. Individuals with a lower ω-3 fatty acid status at baseline benefited from folic acid treatment, while individuals with a higher ω-3 fatty acid status did not. The results potentially explain the inconsistency in outcomes of B-vitamin supplementation trials and emphasize the importance of a personalized approach. This trial was registered at clinicaltrials.gov as NCT00110604.