This study investigated the effects of docosahexaenoic acid (DHA)-rich phospholipid supplementation on behavior, electroretinogram and phospholipid fatty acid (PUFA) composition in selected brain regions and retina in old mice. Two groups of mice were fed a semisynthetic balanced diet or a diet deficient in alpha-linolenic acid. At the age of 8 months, half of each diet group was supplemented with DHA. In the open field, no differences in motor or exploratory activities were observed between the four diet groups. In the light/dark test of anxiety, the time spent in the light compartment was significantly higher in both supplemented groups than in control and deficient groups. Learning performance in the Morris water maze was significantly impaired in deficient old mice, but was completely restored by the phospholipid supplementation. The electroretinogram showed a significant alteration of a- and b-wave amplitudes in control compared to deficient mice. Phospholipid supplementation induced a significant increase of b-wave amplitude in both control and deficient groups and restored normal fatty acid composition in brain regions and retina in deficient mice. DHA-rich phospholipids may improve learning ability, visual function and reverse biochemical modifications in old mice fed an n-3 polyunsaturated fatty acid-deficient diet; they also may improve visual function in old mice fed a balanced diet.