Twenty mature horses (455 to 457 kg and 5 to 10 yr) were utilized in a randomized complete block design to evaluate incorporation of α-linolenic acid into plasma and the effects of supplementation on performance variables in mature horses. Horses were blocked by BW, age, and sex and randomly assigned to treatment within block for a 112 d trial. Dietary treatments included control (no α-linolenic acid; CON) or 150 mg/kg BW/d α-linolenic acid (ALA) derived from a flaxseed oil (TRT; Clear Valley Omega 3 Oil; Cargill, Inc., Eddyville, IA). Diets consisted of CON horses (n = 10) fed 0.25% BW (as-fed) concentrate only or TRT horses fed (n = 10) the same concentrate with additional ALA offered at 12 h intervals. Horses were housed by block and maintained in adjacent dry lots with ad libitium access to coastal Bermudagrass (Cynodon dactylon) hay. Body weight and BCS were obtained every 14 d and concentrate was adjusted accordingly. Rump fat (RF), longissimus dorsi area (LD), and longissimus dorsi fat thickness (LDF) were obtained every 28 d via ultrasonography. Blood samples were collected on d 0, 56, and 112 to determine plasma fatty acid concentrations by gas chromatography. Data were analyzed using the PROC MIXED procedure of SAS. Fatty acid levels in the oil supplement remained stable over the course of the trial. Plasma concentrations of ALA were greater (P ≤ 0.01), and plasma levels of arachidonic acid were lower (P ≤ 0.01) in TRT compared with CON beginning at d 56 to d 112 of the study. Body weight and BCS were not influenced (P = 0.96 and P = 0.14, respectively) by dietary treatment; however, all horses gained BW and BCS throughout the trial (P ≤ 0.01). Rump fat, LD and LDF were not influenced (P ≥ 0.54) by dietary supplementation although, performance variables increased (P ≤ 0.01) over time across treatments. These results indicate that this source of ALA is stable and will incorporate into circulation through targeted supplementation; however, further studies are needed to fully elucidate effects of dietary ALA supplementation to mature horses.