Twelve mature and six 2-year-old Arabian horses were used to determine the effect of dietary long-chain polyunsaturated omega-3 fatty acid supplementation on plasma fatty acids and lameness. Lameness scores and stride lengths were measured on day 0. Horses were striated and pair-matched according to age, gender, stride length and, for mature horses, lameness score, and each horse was fed either a treatment diet containing 5.95 g of stabilized omega-3 fatty acids plus a fat carrier (FA), for a total of 19.4 g fat, or a control diet containing 49 g of corn oil (CO) for 75 days. Horses were exercised 5 d week− 1, and blood samples were drawn and body weights recorded on days 0, 25, 50 and 75. Lameness scores and stride lengths were recorded again on day 75. Total plasma omega-3 fatty acid concentrations were higher on all days in FA horses than in CO horses. Total plasma omega-6 fatty acids increased from days 0 to 25, remained elevated through day 50 and returned to baseline on day 75 in all horses. The ratio of plasma omega-6:omega-3 fatty acids was lower in FA horses. Horses on FA had increased plasma docosahexaenoic acid (DHA) on days 25, 50 and 75. No difference in walk stride length was noted; however, FA horses tended to have a longer trot stride after supplementation when compared with CO horses. No differences were seen in prostaglandin E2 (PGE2) metabolite or tumour necrosis factor-α as measured in blood serum. In summary, supplementing omega-3 fatty acids increases plasma DHA, although there was no overall increase in omega-3 in FA horses. While a trend to increase trot stride length was seen, no differences in lameness scores between treatments were noted.