Omega-3 and n-6 fatty acids are biosynthetic precursors to lipid mediators with antinociceptive and pronociceptive properties. We conducted a randomized, single-blinded, parallel-group clinical trial to assess clinical and biochemical effects of targeted alteration in dietary n-3 and n-6 fatty acids for treatment of chronic headaches. After a 4-week preintervention phase, ambulatory patients with chronic daily headache undergoing usual care were randomized to 1 of 2 intensive, food-based 12-week dietary interventions: a high n-3 plus low n-6 (H3-L6) intervention, or a low n-6 (L6) intervention. Clinical outcomes included the Headache Impact Test (HIT-6, primary clinical outcome), Headache Days per month, and Headache Hours per day. Biochemical outcomes included the erythrocyte n-6 in highly unsaturated fatty acids (HUFA) score (primary biochemical outcome) and bioactive n-3 and n-6 derivatives. Fifty-six of 67 patients completed the intervention. Both groups achieved targeted intakes of n-3 and n-6 fatty acids. In intention-to-treat analysis, the H3-L6 intervention produced significantly greater improvement in the HIT-6 score (-7.5 vs -2.1; P <0.001) and the number of Headache Days per month (-8.8 vs -4.0; P = 0.02), compared to the L6 group. The H3-L6 intervention also produced significantly greater reductions in Headache Hours per day (-4.6 vs -1.2; P = 0.01) and the n-6 in HUFA score (-21.0 vs -4.0%; P <0.001), and greater increases in antinociceptive n-3 pathway markers 18-hydroxy-eicosapentaenoic acid (+118.4 vs +61.1%; P <0.001) and 17-hydroxy-docosahexaenoic acid (+170.2 vs +27.2; P <0.001). A dietary intervention increasing n-3 and reducing n-6 fatty acids reduced headache pain, altered antinociceptive lipid mediators, and improved quality-of-life in this population. © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.