Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. Recent evidence from animal studies suggests that supplementation with omega-3 fatty acids (O3FA) improves functional outcomes following focal neural injury. The purpose of this study is to determine the benefits of DHA supplementation following diffuse axonal injury in rats. Four groups of 10 adult male Sprague-Dawley rats were subjected to an impact acceleration injury and then received 30 days of supplementation with either 10 mg/kg/d or 40 mg/kg/d of docosahexaenoic acid (DHA). Serum fatty acid levels were determined from the isolated plasma phospholipids prior to injury and at the end of the 30 days of DHA supplementation. Following sacrifice, brainstem white matter tracts underwent fluorescent immunohistochemical processing for labeling of β-amyloid precursor protein (APP), a marker of axonal injury. Dietary supplementation with either 10 mg/kg/d or 40 mg/kg/d of DHA for 30 days results in significantly (p < 0.05) increased DHA serum levels of 123% and 175% over baseline, respectively. Immunohistochemical analysis reveals significantly (p < 0.05) decreased numbers of APP-positive axons in animals receiving dietary supplementation with DHA, 26.1 (SD 5.3) for 10 mg/kg/d, and 19.6 (SD 4.7) for 40 mg/kg/d axons per mm(2), versus 147.7 (SD 7.1) axons in unsupplemented animals. Sham-injured animals had 6.4 (SD 13.9) APP positive axons per mm(2). Dietary supplementation with DHA increases serum levels in a dose-dependent manner. DHA supplementation significantly reduces the number of APP-positive axons at 30 days post-injury, to levels similar to seen those in uninjured animals. DHA is safe, affordable, and readily available worldwide to potentially reduce the burden of TBI.